Circadian clock mechanism driving mammalian photoperiodism.

The annual photoperiod cycle provides the critical environmental cue synchronizing rhythms of life in seasonal habitats. In 1936, Bünning proposed a circadian-based coincidence timer for photoperiodic synchronization in plants. Formal studies support the universality of this so-called coincidence timer, but we lack understanding of the mechanisms involved. Here we show in mammals that long photoperiods induce the circadian transcription factor BMAL2, in the pars tuberalis of the pituitary, and triggers summer biology through the eyes absent/thyrotrophin (EYA3/TSH) pathway. Conversely, long-duration melatonin signals on short photoperiods induce circadian repressors including DEC1, suppressing BMAL2 and the EYA3/TSH pathway, triggering winter biology. These actions are associated with progressive genome-wide changes in chromatin state, elaborating the effect of the circadian coincidence timer. Hence, circadian clock-pituitary epigenetic pathway interactions form the basis of the mammalian coincidence timer mechanism. Our results constitute a blueprint for circadian-based seasonal timekeeping in vertebrates.

Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action 'DSDnet' and European Reference Network on Rare Endocrine Conditions.

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

Prevalence of endocrine and genetic abnormalities in boys evaluated systematically for a disorder of sex development.

STUDY QUESTION: What is the likelihood of identifying genetic or endocrine abnormalities in a group of boys with 46, XY who present to a specialist clinic with a suspected disorder of sex development (DSD)? SUMMARY ANSWER: An endocrine abnormality of the gonadal axis may be present in a quarter of cases and copy number variants (CNVs) or single gene variants may be present in about half of the cases. WHAT IS KNOWN ALREADY: Evaluation of 46, XY DSD requires a combination of endocrine and genetic tests but the prevalence of these abnormalities in a sufficiently large group of boys presenting to one specialist multidisciplinary service is unclear. STUDY, DESIGN, SIZE, DURATION: This study was a retrospective review of investigations performed on 122 boys. PARTICIPANTS/MATERIALS, SETTING, METHODS: All boys who attended the Glasgow DSD clinic, between 2010 and 2015 were included in the study. The median external masculinization score (EMS) of this group was 9 (range 1-11). Details of phenotype, endocrine and genetic investigations were obtained from case records. MAIN RESULTS AND THE ROLE OF CHANCE: An endocrine abnormality of gonadal function was present in 28 (23%) with a median EMS of 8.3 (1-10.5) whilst the median EMS of boys with normal endocrine investigations was 9 (1.5-11) (P = 0.03). Endocrine abnormalities included a disorder of gonadal development in 19 (16%), LH deficiency in 5 (4%) and a disorder of androgen synthesis in 4 (3%) boys. Of 43 cases who had array-comparative genomic hybridization (array-CGH), CNVs were reported in 13 (30%) with a median EMS of 8.5 (1.5-11). Candidate gene analysis using a limited seven-gene panel in 64 boys identified variants in 9 (14%) with a median EMS of 8 (1-9). Of the 21 boys with a genetic abnormality, 11 (52%) had normal endocrine investigations. LIMITATIONS, REASONS FOR CAUTION: A selection bias for performing array-CGH in cases with multiple congenital malformations may have led to a high yield of CNVs. It is also possible that the yield of single gene variants may have been higher than reported if the investigators had used a more extended gene panel. WIDER IMPLICATIONS OF THE FINDINGS: The lack of a clear association between the extent of under-masculinization and presence of endocrine and genetic abnormalities suggests a role for parallel endocrine and genetic investigations in cases of suspected XY DSD. STUDY FUNDING/COMPETING INTEREST(S): RN was supported by the James Paterson Bursary and the Glasgow Children's Hospital Charity Summer Scholarship. SFA, RM and EST are supported by a Scottish Executive Health Department grant 74250/1 for the Scottish Genomes Partnership. EST is also supported by MRC/EPSRC Molecular Pathology Node and Wellcome Trust ISSF funding. There are no conflicts of interest. TRIAL REGISTRATION NUMBER: None.

Frequency and aetiology of hypercalcaemia.

BACKGROUND: Hypercalcaemia is rare in children and may present with characteristic signs/symptoms or coincidentally following investigations for a variety of non-specific conditions. The aetiologies of childhood hypercalcaemia are diverse. Untreated sustained hypercalcaemia has serious clinical consequences. However there is limited data regarding the true frequency and aetiologies of childhood hypercalcaemia. AIM: To determine the frequency of severe childhood hypercalcaemia in routine clinical practice. METHODS: The laboratory database was searched for all children (0-17 years) with severe hypercalcaemia defined as non-adjusted ≥2.90 mmol/L from 2007-2012. Hypercalcaemia was categorised as either transient (1 day) or sustained (≥2 consecutive days). Retrospective analysis of all cases of sustained severe hypercalcaemia was performed to identify the underlying aetiology. RESULTS: Over the 5 year period, 206 children were identified as severely hypercalcaemic ≥2.90 mmol/L (0.3% all 61,380 calcium requests). Of these 131 (63.3%) children were classified as having sustained hypercalcaemia. The frequency of severe hypercalcaemia was highest in neonates (42% of sustained cases) and was inversely related to age. Sepsis was the most common aetiology (24%), particularly in neonates where it accounted for 41% of all causes of neonatal hypercalcaemia. Endocrine aetiologies included congenital adrenal hyperplasia (2 cases), fat necrosis (1), Addison's disease (2). A genetic cause was identified in 3 children (2 familial hypocalciuria hypercalcaemia, 1 Williams syndrome). CONCLUSIONS: Sustained hypercalcaemia affects 1 in 500 children in a general hospital setting. The frequency was highest in neonates and underlying aetiology differed markedly with age. All children with sustained hypercalcaemia require thorough investigation to determine the underlying aetiology to ensure appropriate management.

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA).

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.

The concordance between serum anti-Mullerian hormone and testosterone concentrations depends on duration of hCG stimulation in boys undergoing investigation of gonadal function.

BACKGROUND: In boys undergoing investigation of gonadal function, the relationship between a single measurement of serum anti-Mullerian hormone (AMH) and hCG stimulated serum testosterone is unclear. AIM: The aim of the study was to assess concordance between serum AMH and testosterone concentrations following hCG stimulation of two different durations. METHODS: Samples from 284 children (M : F, 154 : 130) with a median age of 8 years (10th, 90th centiles, 0.25, 14) were used to establish an AMH reference range. Clinical data were reviewed in boys undergoing investigation of gonadal function and who had an AMH measurement and a hCG stimulated (3-day or 3-week) (n = 26) testosterone. Of these 26 boys, 11 had combined genital anomalies, whereas the rest had conditions such as isolated hypospadias, undescended testes or microphallus. Normal testosterone response to hCG stimulation was defined as a level greater than 3.5 nmol at day 4 and 9.5 nmol/l at day 22. RESULTS: In the reference group, the 5th centile AMH for boys below 1 year was 215 pmol/l and between 1 and 8 years 180 pmol/l. The 95th centile for girls for these respective age groups was 30 pmol/l and 25 pmol/l. In those cases where serum testosterone concentrations were available at day 1, day 4 and day 22 of the 3 week-hCG test, five cases had a normal serum testosterone at day 4 and three cases only showed such a response by day 22. In those where serum AMH was less than 180 pmol/l, a poor testosterone response of less than 3.5 nmol was observed in approximately seven of eight (88%) cases with a 3-day hCG stimulation test or the 3-week test. An AMH of greater than 180 pmol/l was associated with a normal testosterone response at day 4 in 10 out of 15 (67%) cases and at day 22 in eight of 11 (73%) cases. However, a low serum testosterone concentration of less than 3.5 nmol after the 3-day hCG test was only associated with a likelihood of a low AMH in three of eight (37%) cases. With the 3-week hCG test, a low day 22 testosterone of 9.5 mmol/l or less was associated with a low AMH of 180 pmol/l or less in four of seven (57%) cases. CONCLUSION: In boys undergoing investigation of gonadal function, the concordance between AMH and testosterone is better at day 22 than day 4. A normal AMH may provide useful information on overall testicular function but does not exclude the need for an hCG stimulation test.

N-terminal pro-B-type natriuretic peptide levels and early outcome after cardiac surgery: a prospective cohort study.

BACKGROUND: N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a powerful predictor of cardiovascular outcome in many circumstances. There are, however, limited data regarding the utility of NT-proBNP or BNP levels in patients undergoing cardiac surgery. The current study assesses the ability of NT-proBNP to predict early outcome in this setting. METHODS: One thousand and ten patients undergoing non-emergent cardiac surgery were recruited prospectively. Baseline clinical details were obtained and the European System for Cardiac Operative Risk Evaluation (EuroSCORE) and Parsonnet score were calculated. Preoperative NT-proBNP levels were measured using the Roche Elecsys assay. The primary endpoint was 30 day mortality. RESULTS: Median NT-proBNP levels were 624 ng litre(-1) among patients who died within 30 days of surgery (n=29), compared with 279 ng litre(-1) in survivors [odds ratio (OR) 1.03 per 250 ng litre(-1), 95% confidence interval 1.01-1.05, P=0.001). NT-proBNP levels remained predictors of 30 day mortality in models including either the additive EuroSCORE (OR 1.03 per 250 ng litre(-1), P=0.01), the logistic EuroSCORE (OR 1.03 per 250 ng litre(-1), P=0.004), or the Parsonnet score (OR 1.02 per 250 ng litre(-1), P=0.04). Levels of NT-proBNP were also predictors of prolonged (>1 day) stay in the intensive care unit (OR 1.03 per 250 ng litre(-1), P<0.001) and of a hospital stay >1 week (OR 1.07 per 250 ng litre(-1), P<0.001). They remained predictive of these outcomes in regression models that included either the EuroSCORE or the Parsonnet score and in a model that included all study variables. CONCLUSIONS: NT-proBNP levels predict early outcome after cardiac surgery. Their prognostic utility is modest-but is independent of traditional indicators and conventional risk prediction scores.

Bone morphogenetic protein-4 interacts with activin and GnRH to modulate gonadotrophin secretion in LbetaT2 gonadotrophs.

We have shown previously that, in sheep primary pituitary cells, bone morphogenetic proteins (BMP)-4 inhibits FSHbeta mRNA expression and FSH release. In contrast, in mouse LbetaT2 gonadotrophs, others have shown a stimulatory effect of BMPs on basal or activin-stimulated FSHbeta promoter-driven transcription. As a species comparison with our previous results, we used LbetaT2 cells to investigate the effects of BMP-4 on gonadotrophin mRNA and secretion modulated by activin and GnRH. BMP-4 alone had no effect on FSH production, but enhanced the activin+GnRH-induced stimulation of FSHbeta mRNA and FSH secretion, without any effect on follistatin mRNA. BMP-4 reduced LHbeta mRNA up-regulation in response to GnRH (+/-activin) and decreased GnRH receptor expression, which would favour FSH, rather than LH, synthesis and secretion. In contrast to sheep pituitary gonadotrophs, which express only BMP receptor types IA (BMPRIA) and II (BMPRII), LbetaT2 cells also express BMPRIB. Smad1/5 phosphorylation induced by BMP-4, indicating activation of BMP signalling, was the same whether BMP-4 was used alone or combined with activin+/-GnRH. We hypothesized that activin and/or GnRH pathways may be modulated by BMP-4, but neither the activin-stimulated phosphorylation of Smad2/3 nor the GnRH-induced ERK1/2 or cAMP response element-binding phosphorylation were modified. However, the GnRH-induced activation of p38 MAPK was decreased by BMP-4. This was associated with increased FSHbeta mRNA levels and FSH secretion, but decreased LHbeta mRNA levels. These results confirm 1. BMPs as important modulators of activin and/or GnRH-stimulated gonadotrophin synthesis and release and 2. important species differences in these effects, which could relate to differences in BMP receptor expression in gonadotrophs.

The utility of B-type natriuretic peptide in predicting postoperative cardiac events and mortality in patients undergoing major emergency non-cardiac surgery.

B-type natriuretic peptide (BNP) levels predict cardiovascular risk in several settings. We hypothesised that they would identify individuals at increased risk of complications and mortality following major emergency non-cardiac surgery. Forty patients were studied with a primary end-point of a new postoperative cardiac event, and/or development of significant ECG changes, and/or cardiac death. The main secondary outcome was all-cause mortality at 6 months. Pre-operative BNP levels were higher in 11 patients who suffered a new postoperative cardiac event (p = 0.001) and predicted this outcome with an area under the receiver operating characteristic curve of 0.85 (CI = 0.72-0.98, p = 0.001). A pre-operative BNP value > 170 pg x ml(-1) has a sensitivity of 82% and a specificity of 79% for the primary end-point. In this small study, pre-operative BNP levels identify patients undergoing major emergency non-cardiac surgery who are at increased risk of early postoperative cardiac events. Larger studies are required to confirm these data.

Bilateral vagotomy or atropine pre-treatment reduces experimental diesel-soot induced lung inflammation.

To investigate the role of the vagus nerve in acute inflammatory and cardiorespiratory responses to diesel particulate (DP) in the rat airway, we measured changes in respiration, blood pressure and neutrophils in lungs of urethane anesthetized Wistar rats 6-h post-instillation of DP (500 microg) and studied the effect of mid-cervical vagotomy or atropine (1 mg kg(-1)) pre-treatment. In conscious rats, we investigated DP, with and without atropine pre-treatment. DP increased neutrophil level in BAL (bronchoalveolar lavage) fluid from intact anesthetized rats to 2.5+/-0.7x10(6) cells (n=8), compared with saline instillation (0.3+/-0.1x10(6), n=7; P<0.05). Vagotomy reduced DP neutrophilia to 0.8+/-0.2x10(6) cells (n=8; P<0.05 vs. intact); atropine reduced DP-induced neutrophilia to 0.3+/-0.2x10(6) (n=4; P<0.05). In conscious rats, DP neutrophilia of 8.5+/-1.8x10(6), n=4, was reduced by pre-treatment with atropine to 2.2+/-1.2x10(6) cells, n=3. Hyperventilation occurred 6 h after DP in anesthetized rats with intact vagi, but not in bilaterally vagotomized or atropine pre-treated animals and was abolished by vagotomy (P<0.05, paired test). There were no significant differences in the other variables (mean blood pressure, heart rate and heart rate variability) measured before and 360 min after DP. In conclusion, DP activates a pro-inflammatory vago-vagal reflex which is reduced by atropine. Muscarinic ACh receptors in the rat lung are involved in DP-induced neutrophilia, and hence muscarinic antagonists may reduce airway and/or cardiovascular inflammation evoked by inhaled atmospheric DP in susceptible individuals.

Fetal gonadotrope cell origin of FSH-secreting pituitary adenoma - insight into human pituitary tumour pathogenesis.

OBJECTIVE: The pathogenesis of human pituitary adenomas remains unclear, but we report a case of FSH-secreting pituitary adenoma whose monohormonal phenotype suggests it was of fetal origin. PATIENTS: A 28-year-old woman presented with abdominal discomfort and irregular menses, enlarged multicystic ovaries and elevated serum oestradiol, with sustained high-normal FSH and low LH levels. MEASUREMENTS: Endocrine studies were performed before and after curative surgery, with assessment of tumour hormone secretion in vitro, and immunostaining of tumour tissue for a series of gonadotrope proteins. RESULTS: Immunocytochemistry showed that tumour cells were monohormonal for FSH. Normal components of gonadotrope signalling pathways were expressed, including oestrogen receptor-alpha, activin receptors, secretogranin-II and chromogranin-A. beta-glycan, the putative inhibin coreceptor, was absent. Tumour culture in vitro confirmed secretion of FSH with minimal LH, that was unsuppressed by oestradiol or inhibin-A. Human fetal pituitary tissue contained FSH-only cells at 18 weeks gestation, whereas normal adult pituitary tissue contained only bihormonal gonadotropes. CONCLUSIONS: We propose that this pituitary adenoma represents an indolent tumour of monohormonal fetal gonadotrope cells that originated early in gestation. Pituitary tumours may therefore arise from abnormal persistence of fetal cell types, with extremely slow growth over many years until reaching a size threshold to generate an endocrine syndrome. Understanding fetal pituitary architecture and function may be more informative for new insights into pituitary tumour pathogenesis than classical theories of cancer biology that invoke unrestrained cell proliferation.

Soluble transition metals in welding fumes cause inflammation via activation of NF-kappaB and AP-1.

We previously reported that the molecular pro-inflammatory effects of welding fumes in vitro were caused by soluble transition metals via an oxidative stress-mediated mechanism. Herein, we tested the hypothesis that transition metals in welding fume drive the in vivo inflammatory response caused by welding fume. Rats were instilled with either whole, soluble extract or washed welding fume particulates or soluble extracts pre-treated with a transition metal chelator. Markers of pulmonary inflammation were measured in the bronchoalveolar lavage fluid (BALF) and nuclear translocation of transcription factor was assessed in BAL cells by electrophoretic mobility shift assay. Instillation of either whole or soluble fractions of welding fume caused a significant influx of inflammatory cells and other markers of inflammation in the BALF 24 h later. MIP-2 protein in BALF and nuclear translocation of NF-kappaB and AP-1 were significantly greater following instillation of whole and soluble fractions than in saline-instilled lungs. Chelation of the soluble fraction, to remove transition metals, abolished the ability to cause inflammation, MIP-2 increase or transcription factor translocation to the nucleus. Instillation of washed particulates alone caused no significant change in any end-point compared to saline. This study demonstrates that soluble transition metals present in welding fumes cause inflammation via activation of the redox-sensitive transcription factors NF-kappaB and AP-1 and confirms the validity of utilising in vitro models to assess inflammatory responses to such particles.

Differential secretion of gonadotrophins: investigation of the role of secretogranin II and chromogranin A in the release of LH and FSH in LbetaT2 cells.

This study investigated the role of the secretory granule proteins, secretogranin II (SgII) and chromogranin A (CgA), in the differential secretion of FSH and LH from LbetaT2 mouse gonadotroph cells. Exogenous activin, which synergises with GnRH, is essential for the release of FSH from these cells, but also has stimulatory effects on LH and enhances GnRH-induced LH secretion. Two experiments are reported. In experiment 1, cultures were supplemented with activin (0-50 ng/ml), with and without a daily 1 h treatment of 10 nM GnRH, for 3 days. Protein secretion and mRNA levels were measured. In experiment 2, cells were treated with activin (50 ng/ml) alone, a daily 1 h treatment of 10 nM GnRH, or a combination of both for 6 days. In addition, cells exposed to activin+GnRH for 3 days were subsequently left untreated or given activin or GnRH alone for a further 3 days for comparison with cells maintained in activin+GnRH for 6 days. Protein secretion, intracellular protein and mRNA levels were measured. FSH secretion was stimulated, dose dependently, by activin and this effect increased synergistically in the presence of GnRH. The close correlation between secreted and intracellular FSH and FSHbeta mRNA levels was maintained in cells that had undergone treatment withdrawal after previous exposure to activin+GnRH, but there was no correlation between FSH and the granins. These results are consistent with the view that FSH released in response to activin/GnRH is constitutively secreted via a granin-independent pathway. SgII secretion mirrored the GnRH-induced secretion of LH, but was unaffected by activin, which stimulated LH secretion and had a detrimental effect on CgA mRNA transcription. This confirms previous observations that the LH released in response to GnRH is co-released with SgII via a regulated, granin-dependent pathway, and, in addition, suggests that activin may stimulate LH secretion through a constitutive, granin-independent pathway.

No evidence for pituitary priming to gonadotropin-releasing hormone in relation to luteinizing hormone (LH) secretion prior to the preovulatory LH surge in ewes.

The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 microg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 microg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHbeta mRNA levels were similar between groups. Mean FSHbeta mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH(+ve)/secretogranin II(-ve) (SgII(-ve)) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH(+ve)/SgII(-ve) granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.

The differential secretion of FSH and LH: regulation through genes, feedback and packaging.

While the role of oestradiol and progesterone in the control of GnRH pulsatile secretion and generation of the preovulatory GnRH surge to induce release of the LH surge has been fully investigated, less attention has been given to changes in the pituitary gland that may sensitize gonadotrophs to switch from pulsatile release to surge release of LH, in particular. Furthermore, in the follicular phase while pulsatile secretion of LH is maximal, FSH secretion is reduced, yet both hormones are produced by the same gonadotrophs. The mechanisms whereby this differential release can occur are still unclear. The main regulator of FSH secretion is through the negative feedback effects of oestradiol and inhibin, which directly affect FSHbeta mRNA content and subsequent synthesis of FSH. FSH is then released predominantly via a constitutive pathway and the amount released is closely related to the rate of synthesis. In contrast, while basal LH secretion occurs via a constitutive pathway, the principal release of LH through pulsatile secretion is through the regulated pathway with GnRH stimulating the release of pre-synthesized LH contained in storage granules without significant changes in LHbeta mRNA. Secretogranin II (SgII) is associated with LH in these electron-dense storage granules and LH-SgII granules appear to be the principal form of granule released in response to GnRH through the regulated pathway. At the time of the preovulatory LH surge, granule movement to the gonadotrope cell membrane abutting a capillary, polarization, appears to play an important part in the priming mechanism for release of LH during the preovulatory LH surge in response to the GnRH surge. As there appears to be limited or no gonadotroph cell division in the adult pituitary gland, each gonadotroph passes through this synthesis and secretion pathway repeatedly through successive oestrous cycles. Packaging of LH and FSH into different secretory granules within the same cell is thus pivotal for the differential secretion of these gonadotrophins.

Influence of steroids and GnRH on biosynthesis and secretion of secretogranin II and chromogranin A in relation to LH release in LbetaT2 gonadotroph cells.

The granin proteins secretogranin II (SgII) and chromogranin A (CgA) are commonly found associated with LH and/or FSH within specialised secretory granules in gonadotroph cells, and it is possible that they play an important role in the differential secretion of the gonadotrophins. In this study we have examined the regulation of the biosynthesis and secretion of SgII and CgA, in relation to LH secretion, in the LbetaT2 mouse pituitary gonadotroph cell line. Three experiments were carried out to investigate the effects of oestradiol (E2) and dexamethasone (Dex) in the presence and absence of GnRH (experiment 1), differing GnRH concentrations (experiment 2) and alterations in GnRH pulse frequency (experiment 3). In experiment 1, exposure to E2, Dex or E2+Dex, either with or without GnRH treatment, resulted in increased LH secretion. Steroids alone had no effect on LHbeta mRNA levels, but in the presence of GnRH LHbeta mRNA levels were increased in Dex- and E2+Dex-treated cells. GnRH receptor (GnRH-R) mRNA levels were up-regulated by Dex and E2+Dex, but were unaffected by GnRH. There were no steroid-induced changes in SgII or CgA mRNA, but increased levels of CgA mRNA were observed after GnRH treatment in cells cultured in the presence of Dex. In experiment 2, increasing concentrations of GnRH resulted in increases in LH secretion that were inversely dose-dependent. No changes in LHbeta, GnRH-R or SgII mRNA levels were observed, but there were dose-dependent increases in CgA mRNA levels. In experiment 3, GnRH was given as either 1 pulse/day or 4 pulses/day for 3 days. Both pulse regimes resulted in increased LH, SgII and CgA secretion compared with controls during the first 15 min pulse on day 3. Exposure to GnRH at 4 pulses/day increased LH and SgII secretion compared with controls during all 4 pulses, but secretion of both proteins was reduced during pulses 2-4 compared with pulse 1. CgA secretion also increased due to GnRH in pulse 1, but was decreased by GnRH treatment during pulse 2, and unchanged by GnRH during pulses 3 and 4. Total daily secretion of LH and SgII from cells given 1 pulse/day of GnRH increased compared with controls on all three treatment days, while total CgA secretion increased in response to GnRH on days 2 and 3 only. Intracellular levels of SgII, but not LH, decreased after GnRH treatment. In contrast, intracellular CgA was increased, but only after 4 pulses/day of GnRH. Levels of LHbeta, but not SgII, mRNA were increased by both pulse regimes, while CgA mRNA levels increased after 1 pulse/day of GnRH. These results indicate that there is a close correlation between the GnRH-stimulated release of LH and SgII from LbetaT2 cells, suggesting that SgII may have an influential role in the regulated secretion of LH, possibly by inducing LH aggregation to facilitate trafficking into secretory granules. CgA secretion does not appear to be closely associated with that of LH, but CgA expression does appear to be regulated by GnRH, which may indicate involvement in the control of LH secretion, possibly by influencing the proportion of LH in the different types of secretory granules.

Promotion of intragranular co-aggregation with LH by enhancement of secretogranin II storage resulted in increased intracellular granule storage in gonadotrophs of GnRH-deprived male mice.

Intracellular associations indicate that granins may play a role in the regulatory mechanisms involved in differential secretion of gonadotrophins. The effect of GnRH on mRNA expression, storage and secretory patterns of granins and gonadotrophins was investigated in male mice. GnRH antiserum (G/A) was injected into mice in the treatment group (n = 15) at 12 h intervals for 2 days and a subset (n = 9) was killed. Buserelin (G/A + B) was administered to the remaining mice (n = 6), which were killed 2 h later; control mice (n = 6) were killed at the onset of the study. LHb mRNA content was lower in G/A and G/A + B mice compared with controls, whereas plasma LH concentrations were higher in G/A + B mice. FSHbeta mRNA content did not change, whereas plasma FSH concentrations were lower in G/A mice compared with controls, and higher in G/A + B mice compared with both G/A and control mice. Secretogranin II (SgII) and CgA mRNA contents were not different between experimental groups. There were more granules per gonadotroph in G/A mice, and considerably fewer after Buserelin treatment. Immunogold labelling of gonadotrophs revealed the presence of LH(+ve)/SgII(+ve) and LH(+ve)/SgII(-ve) granules, and negligible numbers of LH(-ve)/SgII(+ve) granules. Both the numbers of LH(+ve)/SgII(+ve) granules and overall granule antigenicity for SgII were higher in G/A mice compared with controls and G/A + B mice. In contrast, there were fewer LH(+ve)/SgII(-ve) granules per gonadotroph in G/A mice compared with controls. In conclusion, absence of GnRH input to the pituitary gland resulted in preferential storage of SgII and subsequently increased intragranular co-aggregation with LH. Administration of Buserelin to G/A mice resulted in the apparent release of LH(+ve)/SgII(+ve) granules that was reflected by an increase in plasma LH concentrations, indicating that these granules were in the regulated secretory pathway. In contrast, secretion of LH(+ve)/SgII(-ve) granules did not appear to be influenced by the actions of Buserelin and, therefore, may have been destined for constitutive release, possibly to maintain basal plasma LH concentrations.

Manipulating the in vivo mRNA expression profile of FSH beta to resemble that of LH beta does not promote a concomitant increase in intracellular storage of follicle-stimulating hormone.

The beta-subunits of luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) are differentially expressed, and this may contribute to the unique expression and storage patterns of LH and FSH. Therefore, to determine if the in vivo expression profile of FSH beta could be altered to that of LH beta, a truncated ovine FSH beta (oFSH beta) gene, which would encode a mRNA lacking the putative destabilizing 3' untranslated region, was fused downstream of the ovine LH beta (oLH beta) promoter and expressed in transgenic mice. In two independent lines, line 16 and 17, we measured oFSH beta, mouse LH beta (mLHbeta) and mouse FSH beta (mFSH beta) mRNA levels: (i) after castration in males; (ii) after administering inhibin to ovariectomized mice; and (iii) during the oestrous cycle. In each experiment, the expression profile of oFSH beta mRNA mimicked mLH beta and not mFSH beta mRNA. In addition, after actinomycin D treatment of pituitary cultures, while mFSH beta mRNA did decay, there was no measurable decay of the oFSH beta mRNA transcript. These differences increased total FSH beta steady-state mRNA expression levels in male transgenics. However, there was no detectable increase in pituitary FSH by either radioimmunoassay or western blotting analysis of pituitary extracts. Subsequent analysis revealed that pituitary FSH beta in line 16 was heavily glycosylated; in contrast, pituitary FSH beta in line 17 was largely unmodified. These differences in post-translational modification of the beta-subunit, and the lack of intracellular storage, contributed to increased plasma FSH levels and ovulation rate in line 16, but not line 17. In conclusion, the expression profile of oFSH beta mRNA was manipulated to mimic mLH beta mRNA and this increased FSH beta mRNA expression levels, but did not increase storage of FSH. This suggests that, regardless of the levels of synthesis, post-translational sorting preferentially promotes FSH secretion from the pituitary.

Loss of oocytes in Dazl knockout mice results in maintained ovarian steroidogenic function but altered gonadotropin secretion in adult animals.

Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (-/-) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from -/- females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor alpha (ER alpha) and ER beta together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ER beta, inhibin alpha, and betaB-subunits, and -/- mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P < 0.01) lower LH and FSH in intact +/+ and +/- females. However, significantly (P < 0.05) increased plasma inhibin B together with significantly (P < 0.05) lower FSH were observed in the +/- females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the +/- mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle.