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.

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.

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.

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.

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.

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.

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.

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Relationship between gonadotrophin subunit gene expression, gonadotrophin-releasing hormone receptor content and pituitary and plasma gonadotrophin concentrations during the rebound release of FSH after treatment of ewes with bovine follicular fluid during the luteal phase of the cycle.

The modulation of FSH secretion at the beginning and middle of the follicular phase of the cycle represents the key event in the growth and selection of the preovulatory follicle. However, the mechanisms that operate within the pituitary gland to control the increased release of FSH and its subsequent inhibition in vivo remain unclear. Treatment of ewes with bovine follicular fluid (bFF) during the luteal phase has been previously shown to suppress the plasma concentrations of FSH and, following cessation of treatment on day 11, a rebound release of FSH occurs on days 12 and 13. When luteal regression is induced on day 12, this hypersecretion of FSH results in an increase in follicle growth and ovulation rate. To investigate the mechanisms involved in the control of FSH secretion, ewes were treated with twice daily s.c. injections of 5 ml bFF on days 3-11 of the oestrous cycle and luteal regression was induced on day 12 with prostaglandin (PG). The treated ewes and their controls were then killed on day 11 (luteal), or 16 or 32 h after PG and their pituitaries removed and halved. One half was analysed for gonadotrophin and gonadotrophin-releasing hormone (GnRH) receptor content. Total pituitary RNA was extracted from the other half and subjected to Northern analysis using probes for FSH-beta, LH-beta and common alpha subunit. Frequent blood samples were taken and assayed for gonadotrophins. FSH secretion was significantly (P less than 0.01) reduced during bFF treatment throughout the luteal phase and then significantly (P less than 0.01) increased after cessation of treatment, with maximum secretion being reached 18-22h after PG, and then declining towards control values by 32h after PG. A similar pattern of LH secretion was seen after bFF treatment. Pituitary FSH content was significantly (P less than 0.05) reduced by bFF treatment at all stages of the cycle. No difference in the pituitary LH content was seen. The increase in GnRH receptor content after PG in the controls was delayed in the treated animals. Analysis of pituitary mRNA levels revealed that bFF treatment significantly (P less than 0.01) reduced FSH-beta mRNA levels in the luteal phase. Increased levels of FSH-beta, LH-beta and alpha subunit mRNA were seen 16h after PG in the bFF-treated animals, at the time when FSH and LH secretion from the pituitary was near maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

Development and application of a heterologous radioimmunoassay for ovine follicle-stimulating hormone.

A highly specific and sensitive heterologous double antibody radioimmunoassay for ovine follicle-stimulating hormone (oFSH) is described in detail. The assay using a rabbit antiserum to human FSH and either 125I-labelled rat FSH or 125I-labelled oFSH as tracer is specific for FSH. A maximum cross-reaction (B/Bo = 50%) of 0-1% was observed with other ovine, rat or human pituitary hormones or human chorionic gonadotrophin. Serum levels of oFSH in samples collected daily throughout the oestrous cycle showed large individual variations. In five out of nine animals a peak of FSH was observed on the day of oestrus.

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.

Characterization of the ovine LH beta-subunit gene: the promoter is regulated by GnRH and gonadal steroids in transgenic mice.

We have previously demonstrated that 1.9 kb of ovine LH beta promoter fused to bacterial chloramphenicol transferase (CAT) coding sequence is sufficient to target expression of the transgene specifically to the gonadotroph cells of the anterior pituitary in mice with no expression being observed in other tissues. However, it is not known if this region of the ovine LH beta promoter contains the necessary elements that confer transcriptional regulation by gonadal steroids and GnRH. Following gonadectomy, both endogenous pituitary LH and CAT activity significantly (P > 0.001) increased as did plasma LH. This post-gonadectomy increase in CAT, pituitary and plasma LH could be suppressed in females by treatment with oestradiol alone or oestradiol and progesterone, with an additional significant (P < 0.05) reduction in CAT activity being observed in one line following the combined steroid treatment. In castrated males, testosterone suppressed CAT activity in one line. Treatment of transgenic ovariectomized females with oestradiol alone significantly suppressed plasma LH (P < 0.01) with no change in pituitary LH content. There was no difference in pituitary LH between oestradiol-treated ovariectomized transgenic and non-transgenic females. Treatment of intact females from both lines with either GnRH antiserum or agonist demonstrated a decrease in pituitary CAT activity whereas similar treatment in intact males had no effect. While endogenous pituitary LH concentrations were variable, plasma LH was lower in all treated animals irrespective of line, sex or expression of the transgene. In conclusion, these results indicate that (1) the presence of CAT protein is not toxic and does not compromise either endogenous LH synthesis, storage and secretion and (2) the ovine LH beta-CAT gene is regulated in a similar but more variable manner to the endogenous LH beta gene. This may relate to the use of CAT as a reporter where its release is not necessarily related to that of the endogenous hormone whose synthesis, storage and release may differ.

Characterization of the ovine LH beta-subunit gene: the promoter directs gonadotrope-specific expression in transgenic mice.

The alpha- and beta-subunits of the gonadotropin hormones are expressed in the gonadotrope cells of the anterior pituitary. There are no adequate in vitro systems for the analysis of beta-subunit gene expression. In this study, therefore, transgenic mice have been used to investigate the regulation of expression of the ovine luteinizing hormone beta-gene (oLH beta) in vivo. oLH beta was isolated, characterized, and 1.9 kb of the promoter fused to the bacterial reporter chloramphenicol acetyl-transferase (CAT). Three lines of transgenic mice were generated. CAT enzyme was detected in the pituitary of two lines, whereas the third line did not express. Measurement of endogenous luteinizing hormone and follicle stimulating hormone levels in both expressing lines revealed small differences when compared to controls, but these did not affect the fertility of the animals. Immunostaining of the anterior pituitary revealed that the oLH beta CAT transgene was expressed specifically in gonadotrope cells.

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.