Abnormal response of luteinizing hormone beta subunit to thyrotrophin-releasing hormone in patients with non-functioning pituitary adenoma

OBJECTIVE it has been suggested that the response of free β-subunit of LH (LHβ) to TRH Is the most useful in-vivo marker of gonadotroph adenomas in patients with non-functioning pituitary adenomas (NFPA). The aim of the present study was to investigate LHβ secretion in patients with NFPA in whom other markers of gonadotroph adenomas, such as supranormal basal concentrations or responses of intact gonadotrophins to TRH, were absent. DESIGN AND PATIENTS Serum basal levels Of LHβ, LH and FSH were evaluated in 80 patients with NFPA showing normal levels of intact gonadotrophin, 20 with PRL-secreting adenomas, 25 with OH-secreting adenomas and 58 healthy subjects. Moreover, LHβ, LH, FSH and alpha-subunit (α-SU) were evaluated in 27 patients with NFPA In whom intact gonadotrophin responses to TRH were absent, 8 with PRL-oma, 7 with GH-oma and 17 healthy subjects before and 20,30 and 60 minutes after the intravenous administration of either 200 μg TRH or placebo. A response was considered present when serum LHβ increased by at least 50% above basal levels. MEASUREMENTS LHβ was evaluated using a new assay based on the sequestration of the combined and free α-SU by an anti α-SU blotinylated monoclonal antibody (MAb) and the subsequent measurement of the LHβ by an IFMA method employing two MAbs directed towards two different epitopes on LHβ. intact LH and FSH were assayed with an IFMA method and α-SU with an IRMA method. RESULTS in basal conditions, no significant difference in the mean values of LHβ was observed among patients with different types of tumour and normal controls. In 9 of 27 (33%) patients with NFPA, TRH caused an abnormal elevation of serum LHβ (net increase 410 ± 403%, range 71-1300) which was completely dissociated from changes in intact gonadotrophins. Of the 5 patients who had a TRH test repeated after transsphenoidal surgery, abnormal LHβ responses disappeared in 2 and were maintained in 3. Disappearance of LHβ response occurred only in patients in whom improvement of visual field and radiological imaging after adenomectomy was observed. in contrast, in ail patients with pituitary tumours other than NFPA and healthy subjects a response to TRH was absent (net increase ranging from 0 to 23%). immunofluorescence, performed on 14 NFPA removed from patients either responsive or unresponsive to TRH, showed a variable proportion of cells positive for LHβ, without a significant difference between the two groups. CONCLUSIONS These results indicate that measurement of basal LHβ is of poor value in the diagnosis of non-functioning pituitary adenomas and the identification of gonadotroph adenomas among non-functioning pituitary adenomas. Conversely, an abnormal response of free LHβ to TRH occurs in about a third of patients with low/normal basal gonadotrophins unresponsive to TRH stimulation.     

Characteristics of acromegalic patients with a good response to octreotide, a somatostatin analogue

BACKGROUND AND OBJECTIVES In GH-secreting pituitary tumours somatostatin receptor density has been correlated with octreotide responsiveness. Little is known about the other endocrine characteristics of patients with good responses to octreotide. The purpose of this study was to determine the characteristics of these patients. PATIENTS We studied 30 patients with active acromegaly. Five had been treated with either transsphenoidal adenomectomy or conventional radiotherapy without cure of GH excess. DESIGN Patients were divided into good or poor octreotide responders. Patients whose GH level decreased to less than 20% of basal and below 20 mU/l after a subcutaneous injection of 100 μg of octreotide were defined as good octreotide responders. We compared tumour size, basal GH secretory pattern, responses to TRH, GnRH and bromocriptine, and mutation of the α-subunit of stimulatory GTP-binding protein (Gα) between the two groups. MEASUREMENT Tumour size was determined by CT or MRI. Basal GH level was measured hourly between 0800 and 1600 h. GH responses to TRH and GnRH were measured every 30 minutes for 2 hours, and the GH response to oral bromocriptine was measured hourly for 6 hours. The mutation of Gα gene between codons 184 and 251 was examined by direct sequencing using PCR in 5 patients of each group whose tumour tissues were available for the genomic DNA extraction. RESULTS Seventeen patients (57%) were good octreotide responders (group I) and 13 (43%) were poor responders (group II). The mean age, sex, tumour size, tumour grade and the basal OH secretory pattern were not significantly different between the two groups. Group I responded more frequently than group II to TRH (65 vs 25%). Fifty-three per cent of group I patients and none of group II were good bromocriptine responders. Forty-one per cent of group I patients responded to both TRH and bromocriptine. Three of 5 group I tumours had point mutations at codon 201 of the Gα, gene, none of 5 group II tumours had mutations. CGT(Arg) was replaced with TGT(Cys) in two tumours and with AGT(Ser) in one. No mutations were found at codon 227. All three tumours with mutations were from patients responsive to TRH. Two of the three were also good bromocriptine responders. CONCLUSIONS These data Suggest that good octreotide responders are more likely to respond to TRH or bromocriptine. Good octreotide responders may include subgroups with different levels of TRH and dopamine receptor expression. A possible relation between octreotide response and the mutation of Gα gene should be investigated.     

Prevalence of an immunological LH β-subunit variant in a UK population of healthy women and women with polycystic ovary syndrome

OBJECTIVE An Immunological LH β-subunit variant has been described, which is undetectable using monoclonal antibodies directed to the intact LH molecule alone. Subjects have been found homozygous or heterozygous for nucleotide mutations within codons 8 and 15 in the LH β-subunit gene. The prevalence of the variant LH β-subunit has been estimated in a healthy UK population of women of reproductive age and in women with polycystic ovary syndrome (PCOS). The relationship of the variant molecule to the clinical and hormonal parameters of the subjects has been evaluated. DESIGN The control and PCOS subjects were screened for the presence of the mutation by using a ratio of two immunofluorometric assays using monoclonal antibodies (Mab). One assay, not detecting the LH variant, uses a Mab directed to the intact LH molecule and a β-specific Mab. The other assay, detecting both the variant and wildtype LH, uses two β-subunit specific Mabs. The mutations in the LH β-subunit gene were confirmed by restriction fragment length polymorphism. The relationship of the presence of the variant to the clinical and hormonal parameters was assessed by ANOVA. PATIENTS Two hundred and twelve normal ovulatory women, of whom 86 (31%) were obese (body mass Index >25) and 146 (69%) non-obese, and 153 women with PCOS, 115 (75%) obese and 38 (25%) non-obese participated in the study. RESULTS The variant LH was detected In 31 (15%) controls and 32 (21%) PCOS subjects (P=0.124) using specific Mab. Obese PCOS had a higher incidence of the heterozygous LH variant compared to obese controls (odds ratio 2.5, P=0.03), and compared to non-obese PCOS (odds ratio 6.3, P= 0.01). The previously described two mutations in codon 8 and codon 15 were present in all subjects detected to be mutant hetero or homo-zygous by RFLP. There was no relationship between the presence of the variant LH and the clinical and hormonal parameters In the PCOS subjects; however, in the controls the presence of the variant LH was associated with a higher serum total testosterone (P=0.046), oestradiol (P=0.03) and SHBG (P=0.002). CONCLUSIONS The results of this study show that the variant LH β-subunit is a common polymorphism occurring in 15% of a healthy UK population of women. The prevalence was not higher in women with PCOS, though it was over represented in obese women with PCOS. The presence of the variant did not alter the clinical or hormonal expression of the disorder in women with PCOS. Its presence in the controls was however associated with higher serum oestradiol and probably secondary elevation of SHBG and testosterone, suggesting that the variant form of LH may be associated with subtle changes in the function of the hypothalamic-pituitary-gonadal axis.     

SECRETION OF ACTH, LPH AND β-ENDORPHIN FROM HUMAN PITUITARY TUMOURS IN VITRO

Basal and stimulated secretion of immunoreactive ACTH, LPH and β-endorphin from four human pituitary tumours has been studied in vitro using a superfused, isolated cell system. Chromatography of cell secretions under acid-dissociating conditions demonstrated that the human tumour cells released immunoreactive peptides with the elution profiles of α_h (1–39) ACTH, β_h-LPH, γ_h-LPH and β_h-endorphin confirming that β_h-endorphin is secreted by human pituitary tumour cells and is not formed by enzymic cleavage from β_h-LPH in blood. No α- or β_h-MSH, nor any higher molecular weight forms of ACTH or LPH were detected. The cells from all four tumours responded to stimulation with rat stalk-median eminence extract (SME) and synthetic AVP with a concomitant release of ACTH, β-LPH, γ-LPH and γ-endorphin. In contrast to the isolated rat anterior pituitary cells, the pattern of responses to SME and AVP were indistinguishable and the release provoked by rat SME could be accounted for virtually entirely by its vasopressin content. No stimulation of release was observed when the cells were exposed to a variety of biogenic amines. Addition of hydrocortisone to the perfusion buffer of two tumours resulted in a slow inhibition of both basal and stimulated ACTH and LPH release. These data demonstrate that human pituitary tumour tissue from patients with Cushing's disease and Nelson's syndrome can be studied in vitro and that such studies may contribute to a greater understanding of the aetiology of these diseases.     

Glycoprotein hormone α-subunit secretion in prolactinomas and in non-functioning adenomas: relation with the tumour size

OBJECTIVE Free giycoproteln hormone α-subunit plasma levels have been reported to be Increased In glycoprotein hormone-secreting adenomas and In acromegaly, but rarely In prolactinomas and In only two cases of Cushing's disease. The prevalence of elevated plasma α-subunit levels. In patients with non-functioning adenomas is still unclear. In addition, no previous work has described plasma a-subunit levels. In a comprehensive series of adenomas characterized by In-vivo secretion and/or Immunocytochemistry. PATIENTS Thirty-seven patients with definite prolactinomas and 48 with non-functioning tumours characterized by Immunocytachemistry were studied, from a series of 145 consecutive patients Including 33 acromegallcs, 18 patients with glycoprotein hormone-secreting adenomas and 9 with Cushing's disease. MEASUREMENTS Plasma free α-subunit was measured by radioimmunoassay in all patients and In a large sample of normal subjects to establish normal ranges according to sex, age and menstrual status. Tumour volume index was the product in cm³ of length, width and height of the adenoma as assessed by computerized tomography or magnetic resonance Imaging. RESULTS Twelve of the 37 (32%) patients with prolactinomas had Increased plasma a-subunit levels; their tumours were significantly larger with significantly higher plasma PRL levels than those of patients without Increased plasma α-subunit levels(P < 0·02). All prolactlnomas above 50 cm³ were associated with α-subunit secretion, whereas only 6 of 29 smaller tumours were similarly associated. Twelve of the 48 ‘non-functlonlng’ adenomas actually secreted α-subunit in vivo: 8 gonado-trophin-secreting, 2 ‘pure’α-secreting, one with negative Immunocytachemistry and one necrotic adenoma. Their volumes were significantly higher than those of adenomas without increased plasma α-subunit levels (P < 0·04). Plasma α-subunit levels were increased in the 6 patients with TSH-secreting adenomas, 8 of 12 with FSH-secreting adenomas, 11 of 33 acromegalics and none of those with Cushing's disease. CONCLUSION Plasma free α-subunit levels were Increased in 49 of 145 patients (34%). For prolactinomas and ‘non-functioning’ adenomas, α-subunit hypersecretion was seen more often with larger tumours. Half of the cases with Increased free α-subunit. In this series were patients harbouring an adenoma which did not stain for an intact glycoprotein hormone.     

GONADOTROPHIN AND ALPHA SUBUNIT SECRETION BY HUMAN ''FUNCTIONLESS'' PITUITARY ADENOMAS IN CELL CULTURE: LONG TERM EFFECTS OF LUTEINIZING HORMONE RELEASING HORMONE AND THYROTROPHIN RELEASING HORMONE

The long-term effects of LHRH and TRH on gonadotrophin alpha subunit, FSH and LH secretion by cell cultures of four human chromophobic pituitary tumours have been examined. The tumours derived from one male and three female patients who presented because of visual disturbance but had no evident endocrine symptoms. Subsequent serum hormone analysis showed the FSH to be high in the male but low or normal in the post-menopausal females whereas LH levels were low in all patients. In culture, basal hormone secretion could be maintained for periods up to 63 d. All tumours secreted alpha subunit and FSH, but much lower amounts of LH. Addition of LHRH or TRH for a period of 12 to 41 d showed that alpha subunit, FSH and LH secretion were stimulated by LHRH from one tumour, by LHRH and TRH from two tumours. There was always a rapid decline in the LH secretion. The tumour which secreted FSH predominantly was stimulated by TRH. We conclude that human pituitary 'functionless' adenomas can secrete gonadotrophin alpha subunit and FSH in vitro and that secretion can be stimulated during long term releasing hormone experiments. LH secretion, however, cannot be maintained.     

PROLONGED TREATMENT WITH THE GnRH ANALOGUE BUSERELIN DOES NOT AFFECT α-SUBUNIT PRODUCTION BY THE PITUITARY GONADOTROPH

Seven patients with metastatic prostatic cancer were treated with biodegradable implants of the GnRH analogue buserelin and six were treated with buserelin intranasally. After 4-24 weeks of treatment mean serum testosterone concentrations were significantly lower in the patients treated with implants than in those treated intranasally (0.7 vs 1.7nmol/1 respectively; P < 0.01). Also, serum LH concentrations were significantly lower in the group treated with implants. Serum α-subunit concentrations were significantly higher than pretreatment values during buserelin treatment. However, the sum of the concentrations of α-subunit present either as free α-subunit or as a part of LH did not differ significantly from pre-treatment values after 8 weeks or more of buserelin treatment. During buserelin treatment serum LH concentrations measured by radioimmunoassay (RIA) were higher than those measured by immunoradiometric assay (IRMA). Cross-reactivity of α-subunit in the LH RIA accounted for many, but not all, of the observed discrepancies. We conclude that: (1) the principal long-term effect of prolonged buserelin administration on the pituitary gonadotroph is the suppression of LHβ production, while α-subunit production is not affected; (2) the serum concentrations of bioactive LH are better reflected by LH concentrations measured by IRMA than by those measured by RIA. (3) Subcutaneous application of biodegradable buserelin implants is more effective in suppressing serum LH and testosterone concentrations than intranasal buserelin application.     

Expression and Regulation of mRNA for Inhibin/Activin α- and βA-Subunits in the Granulosa Layer of the Two Largest Preovulatory Follicles during the Hen Ovulatory Cycle

In order to understand the role of inhibin and activin in regulating follicular development in the hen, the steady-state mRNA levels of inhibin/activin α- and β_A-subunits in the granulosa layer of the largest (F₁) and second largest (F₂) follicles of the hen were investigated at 4-hr intervals throughout the ovulatory cycle. In addition, because it was hypothesized that luteinizing hormone (LH) regulated β_A-subunit expression, the effect ofin vivoadministration of ovine LH (oLH) on the expression of these subunits during the early- and mid-ovulatory cycle was examined. Northern blot analysis, using³²P-labeled cDNA probes of chicken inhibin/activin α- and β_A-subunits and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal control), revealed that in the F₁follicle, the relative level of β_A-mRNA (n = 3) was low at 23.5 hr and increased (P < 0.05) at 19.5, 15.5, and 11.5 hr before the next predicted ovulation. It then decreased (P < 0.05) at 7.5 hr and was further reduced at 3.5 and 0.5 hr prior to ovulation. In the F₂follicle, β_A-mRNA was maintained at a basal level throughout the sampling period except for a brief increase (P < 0.05) at 0.5 hr before ovulation. In contrast to the β_A-subunit, inhibin α-mRNA was abundantly expressed with no significant variations throughout the ovulatory cycle in either the F₁or the F₂follicle. When oLH was injected at 18 hr before ovulation, 200 but not 100 or 50 μg/kg (n = 3 hens per dose) significantly (P < 0.05) reduced the β_A-mRNA level in the F₁follicle by 2 hr after injection compared to the control (saline). The experiment was repeated at 12 hr before ovulation and both 100 and 200 but not 50 μg/kg oLH significantly (P < 0.05) reduced the expression of β_A-subunit mRNA with no significant difference between 100 and 200 μg/kg oLH. In contrast to the β_A-subunit, inhibin α-subunit mRNA was abundantly expressed and not affected by oLH treatment. Our data indicate that the expression of inhibin/activin β_A- but not α-subunit mRNA is developmentally regulated in the granulosa layer of the two largest follicles during the hen ovulatory cycle. In addition, LH may participate, directly or indirectly, in negative regulation of the β_A-subunit.     

Short stature in homozygous β-thalassaemia is due to disproportionate truncal shortening

BACKGROUND and objective Growth failure In homozygous β-thalassaemia has been recognized for many years, and has persisted despite major treatment advances. In this cross-sectional study, sitting and standing height were measured to determine whether growth failure was disproportionate. DESIGN Patient data were analysed In three age groups, 2-10 years, 11-18 years and 19 and over. Sitting height and sublschlal leg length were also determined In a cohort of parents (n= 19) and normal Greek adolescents (n= 32). PATIENTS AND MEASUREMENTS Of the known 156 patients with homozygous β-thalassaemla In the State of Victoria, 154 (98.7%) attend our Institution. Sitting and standing heights were measured, using Harpenden stadlometers, In 57 of 60 (95%) patients aged 2-18 years and in a random selection of 51 of 89 patients aged 19 and over (57%). Measurements are expressed as mean ± SDS. Other data analysed Included serum concentrations of ferritin, zinc, copper, FSH, LH, oestradlol and testosterone, according to standard laboratory assays, together with pubertal status and bone age in patients aged less than 19 years. RESULTS Standing height standard deviation scores in the 2–10 age group were ?0.687 ± 0.861 (n= 9), in the 11–18 age group were ?1.838 ± 1.413 (n= 48) and In the age group 19 and over were ?1.175 ± 1.126. In Individuals aged 2–10 years, sitting height standard deviation scores (SDS) were ?1.56 ± 1.02, In Individuals 11–18 years were ?3.76 ± 1.51 (n= 48), and In individuals 19 years and over were ?2.77 ± 1.20 (n= 51), compared with sublschlal leg length SDS which were, In Individuals aged 2–10 years 0.214 ± 0.91; In 11-18 years, ?0.063 ± 1.347, and In individuals 19 and over, 0.37 ± 1.18. These data show that the reduction in standing height was the result of truncal shortening. Mean sitting height SDS was significantly lower In children with homozygous β-thalassaemla, compared with their parents (P < 0.001), and in a subgroup of Greek adolescents with homozygousβ-thalassaemia compared with age and sex matched normal Greek adolescents (P < 0.001). No correlation was found between truncal shortening and other clinical and biochemical variables measured. CONCLUSIONS Short stature In our patients with homozygous β-thalassaemla is due to disproportionate truncal shortening. The aetiology of truncal shortening in this patient group is likely to be multifactorial, although hypogonadism and chelation therapy may be contributory factors.     

In vivo secretory potential and the effect of combination therapy with octreotide and cabergoline in patients with clinically non-functioning pituitary adenomas

The secretory capacity, in vivo, of clinically non-functioning pituitary adenomas may possibly predict tumour volume reduction during intensive medical therapy. Ten patients (mean (range) 53 years (26-73)) with clinically non-functioning macroadenomas, > or = 10 mm were studied. The secretory capacity of the adenomas was examined using basal, NaCl and TRH-stimulated LH, FSH and alpha-subunit levels. The effect on tumour volume of 6 months' therapy with the combination of a somatostatin analogue, octreotide 200 microg x 3/day and a dopamine-D2-agonist, cabergoline 0.5 mg x 1/day was studied. The basal LH, FSH and alpha-subunit levels were determined before and during 6 months' therapy with octreotide and cabergoline, and MR scans were used to evaluate tumour volume before and during this period of therapy. Octopus-perimetry was used to examine the visual fields. A reduction in tumour volume (mean +/- SEM (range); 30% +/- 4% (18-46%)) during 6 months of combination therapy with octreotide and cabergoline was recorded only in patients with in vivo secretory potential. Tumour volume was not reduced in four patients: in three of these patients it remained unchanged while in one patient it was observed to have increased (by 14%). Of the six patients with pretherapy secretory capacity, one displayed a very high basal level of alpha-subunit (74 microg/l) despite unmeasurable levels of LH and TSH, and an FSH-level of 1 IU/l. The other five patients presented paradoxical LH, FSH and/or alpha-subunit responses to TRH. A reduction in basal levels of LH, FSH and/or alpha-subunit was observed in all six patients, and the maximum reduction of at least one of the hormonal levels was 66% +/- 7% (50-98%). The basal levels of LH, FSH and alpha-subunit in the 10 patients were (mean +/- SEM (range)), 3.0 IU/l +/- 1.0 (0.0-7.4), 12.7 IU/l +/- 5.0 (0.0-39.0) and 9.0 IU/l +/- 7.0 (0.2-74.0). During six months of therapy with octreotide and cabergoline, the basal levels of LH, FSH and alpha-subunit were reduced by > or = 50% in seven patients - including the six patients with in vivo secretion prior to therapy. No new visual field defects were detected during therapy and no deterioration of existing visual field defects was recorded. The medical therapy was well tolerated. The in vivo basal and TRH-stimulated secretory capacity of LH, FSH and alpha-subunit predicted tumour reduction following intensive medical therapy in all of our patients with non-functioning pituitary adenomas.     

Pituitary adenomas with high and low basal inositol phospholipid turnover; the stimulatory effect of kinins and an association with interleukin-6 secretion

OBJECTIVE We examined basal inositol phospholipid turnover and the response to the kinin, kallidin, in human pituitary adenomas and determined whether or not there was an association between these parameters and interleukin (IL-6) secretion status by the tumours. DESIGN Pituitary adenoma tissue was dispersed and cells were cultured in monolayer for 96 hours. The medium was then removed and assayed for IL-6 and anterior pituitary hormones. The cells were labelled with ³H-myoinositol for 24 hours and then incubated under basal conditions with kallidin and, in some cases, with TRH and GnRH for 60 minutes. Total inositol phosphate accumulation and pituitary hormone secretion were assessed. PATIENTS Tissue was collected from 29 consecutive patients being treated surgically for pituitary adenomas. MEASUREMENTS Total ³H-inositol phosphates, growth hormone, prolactin, LH, FSH, TSH and immunoreactive IL-6. RESULTS Two groups of pituitary adenomas were identified, one with high and one with low basal inositol phospholipid turnover. Kallidin stimulated inositol phosphate accumulation in seven of the 29 adenomas studied. The kallidin-responsive adenomas were associated with high basal phosphoinositide turnover. All seven kallidin-responsive adenomas secreted IL-6. The adenomas studied with high basal inositol phosphate production were also responsive to TRH and in two tumours to GnRH. Kallidin stimulated GH release in one GH-secreting adenoma but had no effect on hormone secretion from any other tumour. CONCLUSION Two groups of pituitary adenomas have been identified with high and low basal inositol phosphoinositide turnover. Phosphoinositide metabolism is readily stimulated by kallidin and TRH in adenomas with high but not low turnover. Kinin-responsive adenomas secreted IL-6 but IL-6 secretor status does not preclude that they will respond to kallidin.     

Luteinizing hormone regulation by sex steroids in men with germinal and Leydig cell tumours

OBJECTIVE We examined the gonadotrophin secretion in patients with increased plasma concentrations of testosterone and oestradiol due to hCG-producing tumours. DESIGN Comparison of plasma gonadotrophin concentrations before and after stimulation by GnRH, in eight men with hCG-producing tumours resulting in Increased testosterone and oestradiol plasma levels, and In 29 men with Leydig cell tumours resulting in increased oestradiol and normal to low testosterone plasma levels. PATIENTS Eight men with hCG-producing tumours (six with testicular tumours, two with extratesticular tumours), 29 men with Leydig cell tumours and 15 normal men. The six men with germinal cell tumours of the testis were studied before and after unilateral orchidectomy. MEASUREMENTS Plasma concentrations of hCG, testosterone and oestradiol were measured before and after intramuscular injection of hCG. LH and FSH were measured before and after intravenous Injection of 100 μg GnRH. RESULTS Plasma LH and FSH concentrations were low In patients with germ cell tumours, who exhibited increased plasma testosterone and oestradiol concentrations, and were normal in patients with Leydig cell tumours, in whom oestradiol only was increased. Plasma LH and FSH were normalized in the five patients with successful (e.g. normal hCG, testosterone and oestradiol) unilateral orchldectomy. Basal plasma testosterone concentrations correlated positively (P <001) with plasma oestradiol concentrations in patients with germ cell tumours and negatively (P <0 01) in patients with Leydig cell tumours. CONCLUSIONS In patients with hCG-secreting germ cell tumours complete suppression of plasma LH and FSH with increased plasma concentrations of both testosterone and oestradiol are often discovered. No such gonadotrophin suppression is found In patients with Leydig cell tumours, but the negative correlation observed between plasma testosterone and oestradiol in these patients suggests a weak negative feedback effect of oestradiol on LH secretion, which cannot be demonstrated by basal LH measurements in plasma.     

Gonadotrophin subunit and GnRH receptor gene expression in the pars distalis of the equine pituitary

In the horse, pronounced changes in fertility occur annually in response to photoperiod. However, the mechanisms regulating gonadotrophin synthesis and release in this species remain unclear. Here, we investigated the expression of gonadotrophin subunits and GnRH receptor (GnRH-R) mRNA in the pituitary glands of Thoroughbred horses during the breeding (BS) and non-breeding (NBS) season. Seasonal effects on the prevalence of gonadotrophs in the pars distalis were also examined. GnRH-R and common α-, LHβ- and FSHβ-subunit mRNA contents were determined by Northern analysis and the prevalence of LH–gonadotrophs assessed by immunohistochemistry in pituitaries from sexually active females (mares) in the BS, and sexually inactive mares in the NBS. These variables were then measured in castrated male horses (geldings). In mares, pituitary content of FSHβ mRNA was significantly higher in the NBS (P < 0.01). Conversely, the content of common α-subunit mRNA was significantly higher during the BS (P < 0.05). In contrast, GnRH-R and LHβ mRNA abundance were unaffected by season. Interestingly, whereas no seasonal effects were apparent on the number of LH–gonadotrophs/field, the proportion of LH cells (in relation to all other cells) was higher in BS than NBS animals (P < 0.05); this resulted from an increased number of non-gonadotroph cells during the NBS (P < 0.05). In geldings, no significant seasonal effects were detected for any of the variables investigated (P > 0.05). These results reveal robust seasonal effects on common α-subunit and FSHβ gene expression in the pituitary of the mare, in the absence of detectable changes in the content of LHβ or GnRH-R mRNA.     

Isolation and Characterization of Two Distinct Gonadotropins from the Pituitary Gland of Mediterranean Yellowtail,Seriola dumerilii(Risso, 1810)

Two gonadotropins, GTH I and GTH II, were isolated and chemically characterized from the pituitary of Mediterranean yellowtail. They were extracted with 35% ethanol–10% ammonium acetate, separated by ion-exchange chromatography on a DE-52 column, and purified by reversed-phase high-performance liquid chromatography on Asahipak C4P-50 and subsequently by gel filtration chromatography on Superdex 75. The molecular weights were estimated at 47 kDa for GTH I and 29 kDa for GTH II by SDS–PAGE and at 49 kDa for GTH I and 42 kDa for GTH II by gel filtration. GTH II was completely dissociated, while GTH I was partially dissociated into α- and β-subunits by treatment with 0.1% trifluoroacetic acid. The complete amino acid sequences of GTH α-, GTH Iβ-, and GTH IIβ-subunits were determined. The GTH α-subunit consisted of 91 amino acid residues. The GTH Iβ and GTH IIβ consisted of 105 and 115 amino acid residues, respectively, and had a 28% sequence identity to each other. They had the highest sequence identity with the respective gonadotropin subunits of bonito, tuna, and striped bass: 81–83% for GTH α, 67–71% for GTH Iβ, and 91–93% for GTH IIβ. The sequence identity of the GTH α-subunit with those of other teleosts and human and bovine LH and FSH was 57–67%. The GTH Iβ-subunit showed a low sequence identity with other known fish GTH Iβs (36–51%) and was more similar to human and bovine FSH βs (34% identity) than to human and bovine LH βs (29% identity). The sequence identity of the GTH IIβ-subunit with those of other teleosts was higher (60–73%), being more similar to LH βs (43% identity) than FSH βs (38% identity). Thus, two distinct gonadotropins, GTH I and GTH II, homologous to mammalian FSH and LH, respectively, are synthetized by M. yellowtail pituitary glands.     

17β-Oestradiol counteracts the formation of the more acidic isoforms of follicle-stimulating hormone and luteinizing hormone after menopause

OBJECTIVE When the gonadotropin levels increase at midcycle, more basic isoforms of FSH and LH appear in the circulation. However, when these gonadotrophins increase at menopause more acidic forms appear. The present study was done to see whether chronic 17β-oestradiol (E₂) administration to post-menopausal women could counteract the formation of the more acidic isoforms after the menopause. DESIGN Serum samples were obtained from 16 postmenopausal women, mean age 70 years (range 63-84 years), 46-169 days after the subcutaneous insertion of a 20-mg E₂-implant. FSH, LH and E₂ in the sera were measured with fluoroimmunoassays. The median charge and the degree of charge heterogeneity of the FSH and LH isoforms were determined for each serum by electrophoresis in 01% agarose suspension. Sera from an age-matched control group were analysed in parallel. RESULTS The E₂ levels in the E₂-treated women were 230–570 pmoI/I, within the range expected during the midluteal phase of the normal menstrual cycle. The mean serum FSH and LH levels were similar to normal follicular phase FSH and LH levels (8 6 and 20 8% respectively of the control group). It was estimated that individual serum specimens from both groups contained 20–30 different isoforms for both FSH and LH. The median charges of the isoforms of FSH and LH were more basic in all the E₂-treated subjects than in their corresponding untreated controls. The mean median charge for FSH was close to the values for the follicular and luteal phases and that for LH close to that for the luteal phase. In some E₂-treated women the isoforms were even more basic with a charge similar to that at the midcycle peak. The degree of charge heterogeneity for the E₂-treated group was significantly (P<0.001) larger than for the controls and similar to that during the normal menstrual cycle. CONCLUSION Chronic E₂ administration to post-menopausal women counteracted the formation of more acidic isoforms of both FSH and LH after the menopause.     

EFFECT OF PROLONGED ADMINISTRATION OF RANITIDINE ON PITUITARY AND THYROID HORMONES, AND THEIR RESPONSE TO SPECIFIC HYPOTHALAMIC-RELEASING FACTORS

We have studied, in a double blind controlled trial, 30 male patients with duodenal ulcer to evaluate the effect of prolonged oral administration of ranitidine (150 mg bd for 4 weeks), a new H2-receptor antagonist, on basal PRL, LH, FSH and TSH concentrations, on their response to specific releasing hormones, and on basal and TRH-stimulated levels of thyroid hormones. Neither the basal levels of PRL, FSH, LH and TSH, nor their response to stimulation with appropriate releasing hormone were affected by ranitidine. Basal concentrations of T4 and its levels after TRH stimulation at 40 min (but not at 20, 60 and 120 min) were lower after ranitidine treatment (P < 0·05); basal and stimulated T3 and rT3 were unaffected. These results could suggest a possible role of histamine in thyroxine regulation but further studies are required.     

Effects of insulin-like growth factor-I on growth hormone and prolactin secretion and cell proliferation of human somatotrophinomas and prolactinomas in vitro

OBJECTIVE IGF-I inhibits GH secretion from normal and some tumorous pituitary tissue, and has been shown to be mitogenic for gonadotrophinoma cells in vitro. It is not known whether IGF-l affects somatotrophinoma cellular proliferation or the secretion of other hormones, such as PRL and α-subunit, which are often co-secreted by these tumours. We have therefore examined the effects of IGF-l on proliferation and hormonal secretion of human somatotrophinomas and prolactinomas in vitro. DESIGN Pituitary adenoma tissue was dispersed to single cells in monolayer culture. The effects of 100 nw IGF-I on GH, PRL and α-subunit secretion were determined over 4-hour and over 4-day periods, and a 4-day dose-response study using 1–100 nM IGF-I was performed on two tumours. Adenoma cell S-phase proliferation was determined after bromodeoxyuridine Incorporation for 1 hour after 4 days, using a double immunostaining method. RESULTS Over 4 hours, 100 nw IGF-I had no effect on GH, PRL or α-subunit secretion in 7 tumours. Over 4 days, 100 nw IGF-I reduced GH secretion In 518 somatotrophinomas (range 17–84%, P < 0·05) compared to controls, with tumours responding to IGF-I having lower basal serum and in-vitro GH levels than tumours unaffected by IGF-I (P < 0·05). There was no effect on α-subunit secretion in any of the three tumours studied. PRL co-secretion was increased In 315 somatotrophinomas compared to control (20, 30 and 37%, P < 0·05), with tumours responding to IGF-I being associated with lower basal serum and in-vitro PRL levels than those tumours unaffected by IGF-I. IGF-I also increased PRL secretion in 2/2 prolactinomas (27 and 32%, P < 0·05) compared with control. GH was inhibited and PRL secretion was stimulated by 1 and 10 nw IGF-I in the two dose-response studies. The proliferative labelling index did not exceed 1·9% in any tumour and no proliferative effect was found with 100 nw IGF-I in any somatotrophinoma. CONCLUSION IGF-I inhibited tumorous GH in 62% and stimulated PRL secretion in 71 % of tumours over 4 days, without affecting α-subunit secretion or being mitogenic for somatotrophinoma cells in vitro. No hormonal effects were observed over short (4-hour) incubations. IGF-I may be a newly recognized factor directly stimulating tumorous PRL secretion.     

THYROID HORMONE, OESTROGEN, AND GLUCOCORTICOID EFFECTS ON TWO DIFFERENT PITUITARY GLYCOPROTEIN HORMONE ALPHA SUBUNIT POOLS

Seven hypothyroid women (six postmenopausal and one premenopausal) were treated with 200 or 300 μg of levo-T4 daily in order to suppress the secretion of TSH and its alpha and beta subunits. After such therapy, serum TSH and beta subunit of TSH (TSH-β) levels were usually not detectable. Serum alpha decreased from a mean value of 4.5 ± 0.8 ng/ml to 2.8 ± 0.4 ng/ml (P > 0.01). Serum LH and FSH levels were unchanged. Oestrogen administration (20 mg diethylstilboestrol daily for 3 or 5 days) to these patients further decreased mean serum alpha to 1.9 ± 0.2 ng/ml (P > 0.005). In addition serum LH, FSH, and the beta subunits of LH and FSH decreased with oestrogen treatment. In separate experiments glucocorticoids (dexamethasone 4 mg every 6 hours for 10 doses) were administered to eight normal individuals, four women with decreased thyroid reserve, and one hyper-thyroid woman. Not only did basal and TRH-stimulated TSH secretion decrease, but secretion of alpha and TSH-β also decreased after dexamethasone administration, with the largest fall in the patients with decreased thyroid reserve. The change in these thirteen patients’ alpha subunit increment after TRH administration was from a mean of 1.6 to 1.0 ng/ml with glucocorticoids; the change in TSH-β was from a mean of 1.1 to 0.7 ng/ml. Serum LH and FSH concentrations were not altered by glucocorticoid administration. These studies have demonstrated two different pools of alpha subunits in the pituitary gland. Thyroid hormone and glucocorticoids decreased secretion of alpha subunits arising from the thyrotroph; oestrogen decreased secretion of alpha subunits arising from the gonadotroph.     

Effect of chronic daily oral administration of 17βoestradiol and norethisterone on the isoforms of serum gonadotrophins in post-menopausal women

OBJECTIVE Chronic treatment with 17βoestradlol (E₂Implants has been found to counteract the formation of more acidic isoforms of the gonadotrophins in post-menopausal women. Oral medication with an oestrogen in combination with a progestagen is a common hormone replacement therapy (HRT) in post-menopausal women. The present study investigated the effect of such a therapy on the concentration and charge of the gonadotrophin isoforms in serum. DESIGN Serum samples were obtained from 20 post-menopausal women, mean age 60 years (range 50–72 years), treated with continuous dally oral medication of 2 mg E₂ combined with 1 mg norethisterone acetate (NETA). FSH, LH and E₂ in the serum was measured with fluoroimmunoassays. The median charge and charge heterogeneity of the FSH and LH isoforms were determined for each serum by electrophoresis in 0·1% agarose suspension. Sera from 20 post-menopausal women without a history of HRT served as controls. The results were compared with those from previous studies on post-menopausal women treated with E₂ Implants and on women with normal menstrual cycles. RESULTS The E₂ level in the oral-E₂+ NETA treated women was 198-610 pmol/l, within the range expected during the mid-luteal phase of the normal menstrual cycle and similar to that of the group of women with an E₂ Implant. The mean LH level was similar to that of the luteal phase of the cycle and significantly lower than that of the controls (P < 0·001), the E₂ Implant group (P < 0·001) and at the follicular phase of the cycle (P < 0·01). The mean FSH level was slmllar to that of the folllcular phase and the E₂ implant group but lower than that of the controls (P < 0·001) and higher than at the luteal phase of the cycle (P < 0·01). The mean values for median charge of both FSH and LH were less acidic than those of the controls (P < 0·001) but more acidic than those for the E₂ Implant group (P < 0·01; P < 0·001) and for different phases of the menstrual cycle (P < 0·05; P < 0·001). The mean degree of charge heterogeneity of FSH was larger (P < 0·01), while that of LH was smaller (P < 0·01), than for the controls. The mean concentrations of SHBG in the oral E₂+ NETA group, the E₂ implant group and the controls were similar. CONCLUSION Chronic oral therapy with 2mg 17β oestradiol combined with 1 mg norethisterone in post-menopausal women efficiently decreased the serum gonadotrophin levels but only partly counteracted the formation of the more acidic isoforms of FSH and LH after menopause. The differences in the charge for both FSH and LH between the E₂ Implant and the oral E₂+ NETA treated groups may be due to the differences in route of administration of E₁ or to the effect of norethisterone or both.