bluuebunny/crossref_metadata_2025 huggingface dataset:
Oestrogen positive feedback stimulates the synthesis of LHRH mRNA in neurones of the rostral diencephalon of the rat
Authors: Rosie R. , Thomson E. , Fink G.
Published: December 2008

Our aim was to determine whether oestrogen administered to ovariectomized rats in a manner which stimulates the release of LHRH also stimulates the synthesis of LHRH mRNA. Adult female Wistar rats were ovariectomized under halothane anaesthesia between 09.00 and 11.00 h of dioestrus, immediately injected with either oil or 10 μg oestradiol benzoate (OB) and then killed by cervical dislocation between 16.00 and 17.00 h of the next day (presumptive pro-oestrus). In-situ hybridization carried out with a 30 mer, 32P-labelled, oligonucleotide probe complementary to LHRH mRNA showed that the concentrations of LHRH mRNA in perikarya in the medial preoptic area, diagonal band and medial septum were significantly greater in OB-compared with oil-treated rats. Plasma LH concentrations were significantly increased in three out of four of the OB-treated rats. These results show for the first time that as well as stimulating LHRH and LH release, oestrogen positive feedback also stimulates the synthesis of LHRH mRNA.

Journal of Endocrinology (1990) 124, 285–289
Measurement of parathyroid hormone-related protein in extracts of fetal parathyroid glands and placental membranes
Authors: Abbas S. K. , Pickard D. W. , Illingworth D. , Storer J. , Purdie D. W. , Moniz C. , Dixit M. , Caple I. W. , Ebeling P. R. , Rodda C. P. , Martin T. J. , Care A. D.
Published: December 2008

A radioimmunoassay based on an antiserum to human parathyroid hormone-related protein PTHrP(1–16) was used with PTHrP(1–34) standard to measure the concentration of immunoreactive PTHrP in extracts of fetal parathyroid glands from lambs and calves and also placental membranes obtained from several species, including man. Dilution curves from these sources were parallel to those obtained for PTHrP(1–34) standard. It was demonstrated that this parallelism was not the result of tracer damage caused by enzymic activity in the tissue extracts. Extracts of human placental membranes were subjected to high-pressure liquid chromatography with a linear acetonitrile gradient. Co-elution of cytochemical biological activity with 125I-labelled PTHrP(1–34) was noted. These results provide further evidence for both the fetal parathyroid glands and the placenta containing material resembling PTHrP which may be responsible for sustaining the activity of the placental calcium pump which maintains the fetus hypercalcaemic relative to its mother.

Journal of Endocrinology (1990) 124, 319–325
Effects of bovine 35 kDa FSH-suppressing protein on FSH and LH in rat pituitary cells in vitro: comparison with bovine 31 kDa inhibin
Authors: Robertson D. M. , Farnworth P. G. , Clarke L. , Jacobsen J. , Cahir N. F. , Burger H. G. , de Kretser D. M.
Published: December 2008

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3–9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC50) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC50 = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC50 values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC50 values =142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum. The pattern of FSP-induced suppression of FSH basal release and cell content and its inhibition of GnRH-stimulated release of FSH and LH was similar to that observed with inhibin, although FSP is 10–20% as potent as inhibin. When inhibin and FSP were assayed either individually or as a 1:1 (v/v) mixture, the experimentally determined biological activity of the mixture did not differ significantly from that predicted from the individual activities.

It was concluded from the similarity in pituitary responses to FSH and inhibin that FSP is a weak inhibin agonist. Its action can be discriminated from that of inhibin by an inhibin antiserum; however, FSP and inhibin may share a common underlying mechanism of action since their effects are additive.

Journal of Endocrinology (1990) 124, 417–423
Initiation of parturition and lactation in the sow: effects of delaying parturition with medroxyprogesterone acetate
Authors: Whitely J. L. , Hartmann P. E. , Willcox D. L. , Bryant-Greenwood G. D. , Greenwood F. C.
Published: December 2008

The synthetic progestagen, medroxyprogesterone acetate (MPA), was administered to sows in late pregnancy with the objective of slightly delaying the time of farrowing and thereby providing more marked associations between hormonal changes and the termination of pregnancy, and the initiation of farrowing and lactation in this species.

MPA was administered orally (140 mg, twice daily) to eight sows in late pregnancy on days 112, 113 and 114 of gestation. Parturition was then induced to occur on day 116 by injecting 200 μg cloprostenol i.m. on day 115 of gestation. The peripartum changes in the plasma concentrations of progesterone, cortisol, oestradiol-17β, relaxin, prolactin, lactose and 13,14-dihydro-15-keto prostaglandin F2α (PGFM) were measured in these sows together with a group of untreated sows. The gestational length for the MPA-treated sows (116·3 ± 0·3 days, mean±s.e.m.) was significantly (P<0·01) greater compared with the untreated sows (114·9 ± 0·3 days). Plasma progesterone declined earlier (P<0·05) with respect to the time of parturition in the treated sows compared with the untreated group. With respect to the timing of parturition, the time at which maximal concentrations of relaxin were attained and the timing of the subsequent decline were earlier in the MPA-treated sows. In both groups of sows, the concentration of relaxin increased before the decline in plasma progesterone. In the untreated sows, the concentration of PGFM increased either slightly before or at the same time as the decline in plasma progesterone, whereas in sows treated with MPA, progesterone concentrations began to decline before any significant increase in the plasma concentration of PGFM. The profiles of cortisol, oestradiol-17β and PGFM were similar in both groups of sows. In both groups of sows, the timing of the initial increase in the concentration of plasma prolactin coincided with a similar rise in plasma lactose (P<0·01). Plasma progesterone either declined earlier or at the same time as the rise in plasma lactose (P<0·01) in the treated group of sows only.

We conclude that since the prepartum changes in the concentration of progesterone and relaxin occurred before significant changes in the concentration of PGFM in the MPA-treated sows, the nature of the luteolytic factor and the mechanism by which it exerts its action remains obscure. The higher concentration of lactose in the mammary secretion at birth in the MPA-treated sows compared with the untreated group suggested that lactogenesis was initiated earlier with respect to parturition following MPA treatment. Furthermore, the administration of MPA to sows in late pregnancy delayed the onset of parturition but did not inhibit lactogenesis.

Journal of Endocrinology (1990) 124, 475–484
Non-sulphated cholecystokinin in human medullary thyroid carcinomas
Authors: Rehfeld J. F. , Johnsen A. H. , Ødum L. , Bardram L. , Schifter S. , Scopsi L.
Published: December 2008

The expression of gastrin/cholecystokinin (CCK) peptides and their precursors was examined in 16 medullary carcinomas of the human thyroid. Measurements with libraries of sequence-specific radioimmunoassays before and after enzymatic cleavage of extracts and chromatographic fractions showed that the carcinomas contained 1·7 pmol carboxyamidated CCK/g tissue (median; range 0·6–21·8 pmol/g), 0·9 pmol glycine-extended precursor/g (median; range < 0·2–2·3 pmol/g) and 2·3 pmol further COOH-terminal-extended proCCK/g (median; range 0·9–6·2 pmol/g). Neither carboxyamidated gastrins nor any progastrins could be measured. Gel and reverse-phase chromatography revealed only small molecular forms, i.e. > 90% of the amidated immunoreactivity eluted like non-sulphated CCK-8 or CCK-7. The results show that human medullary thyroid carcinomas synthesize CCK peptides. The predominance of non-sulphated CCK is unusual. Taken together with earlier observations from dogs and pigs, our results raise the possibility that small non-sulphated CCK peptides modulate thyroid C-cell secretion in an autocrine manner.

Journal of Endocrinology (1990) 124, 501–506
AMYLIN AND AMYLIN-AMIDE LACK AN ACUTE EFFECT ON BLOOD GLUCOSE AND INSULIN
Authors: Ghatei M. A. , Datta H. K. , Zaidi M. , Bretherton-Watt D. , Wimalawansa S. J. , MacIntyre I. , Bloom S. R.
Published: December 2008

Amylin-amide has been implicated in the pathogenesis of type II diabetes due to its proposed inhibitory effect on insulin release from β cells of the pancreatic islets, and on glucose uptake by the skeletal muscle. In experiments with rats and rabbits we failed to demonstrate these anti-insulin actions of amylin and amylin-amide. A single bolus dose of the two peptides (500 pmol) administered i.v. failed to supress plasma insulin levels or to elevate blood glucose levels. The continuous infusion of amylin-amide into rabbits also failed to supress the release of insulin in response to hyperglycaemia produced by an i.v. bolus injection of glucose. These in vivo observations imply that the amylin peptides may not have a primary physiological role in carbohydrate metabolism, but in view of our previous findings, we speculate that the peptide has a more prominent role in calcium homeostasis.
Effect of low temperatures on glucose-induced insulin secretion and glucose metabolism in isolated pancreatic islets of the rat
Authors: Escolar J. C. , Hoo-Paris R. , Castex Ch. , Sutter B. Ch. J.
Published: December 2008

The direct effect of hypothermia on the inhibition of insulin secretion may result from inhibition of the availability of energetic substrates and/or the lack of metabolic signals. In order to verify this hypothesis, the insulin secretion and the main metabolic glucose pathways were measured during the incubation of rat islets. In the presence of 16·7 mmol glucose/l and at 37 °C, insulin secretion was 925 ± 119 μU/2 h per ten islets. With the same experimental conditions, glucose utilization, determined as the formation of 3H2O from [5-3H]glucose was 2225 ±184 pmol/2 h per ten islets, glucose oxidation measured as the formation of 14CO2 from [U-14C]glucose was 673 ± 51 pmol/2 h per ten islets, pentose cycle determined as the formation of 14CO2 from either [1-14C]glucose or [6-14C]glucose was 37 ± 5 pmol/2 h per ten islets; glucose oxidation by the tricarboxilic acid cycle, calculated to be the difference between glucose oxidation and pentose cycle values, was 636 pmol/2 h per ten islets.

Hypothermia highly inhibited glucose-induced insulin secretion and glucose utilization. Inhibition of insulin secretion was partial at 27 °C since it was 2·5 times lower than that at 37 °C, and it was complete at 17 °C. Glucose oxidation in the tricarboxilic acid cycle was markedly inhibited by hypothermia since the inhibition coefficient (Q10) between 37 and 27 °C was 5. In contrast, glucose oxidation in the pentose phosphate shunt was enhanced at 27 °C, reaching 92 ± 17 pmol/2 h per ten islets, and it was inhibited relatively little at 17 °C.

These results suggest that hypothermia markedly inhibits glucose metabolism with the exception of the pentose pathway which could play an important role by inducing the insulin secretion at 27 °C.

Journal of Endocrinology (1990) 125, 45–51
Vasopressin responses to hypoxia in conscious rats: interaction with water restriction
Authors: Griffen S. C. , Raff H.
Published: December 2008

The purpose of this study was to determine the effect of water restriction on the vasopressin response to hypoxia in conscious Long–Evans rats. Rats were prepared with chronic indwelling femoral artery and vein catheters 1 week before experimentation. At 24 h before the first blood sample, the supply of drinking water was maintained ad libitum (water replete) or removed (water deplete). At 24 h, a control blood sample was taken and then normoxia (21% O2) was maintained or hypoxia (10% O2) induced. Additional blood samples were taken at 1, 18 and 24 h. All blood samples (2·5 ml) were simultaneously replaced with donor blood to maintain isovolaemia. Hypoxia led to a very small and transient increase in vasopressin in the water-replete rats. The combination of hypoxia and water restriction led to a greatly augmented vasopressin response at 1 h (60 ± 16 pmol/l); this response was also not sustained. Additional non-cannulated rats were exposed to 24 h of normoxia or hypoxia with or without water available ad libitum and posterior pituitaries were collected after decapitation for measurement of vasopressin content. Water restriction, hypoxia and water restriction plus hypoxia all led to decreased pituitary vasopressin content. We conclude that the vasopressin response to hypoxia in conscious rats is small and transient, and that concomitant water restriction augments the vasopressin response to acute but not chronic hypoxia.

Journal of Endocrinology (1990) 125, 61–66
Hypoinsulinaemia in the lactating rat is caused by a decreased glycaemic stimulus to the pancreas
Authors: Madon R. J. , Ensor D. M. , Flint D. J.
Published: December 2008

An in-vitro perifusion system was devised in order to examine the secretory profiles of isolated islets of Langerhans, derived from different physiological states, when subjected to various stimuli relevant to lactation. Islets from pregnant rats secreted more insulin than did those from virgin animals; however, islets from lactating and virgin animals secreted similar amounts of insulin with all stimuli, including glucose, amino acids, cations and neurotransmitters. When virgin rats were pretreated for 5 days in vivo with GH or prolactin, insulin responses in vitro were unchanged. Cannulation of the hepatic portal vein and inferior vena cava in vivo revealed that both insulin and glucose concentrations were lower in the portal vein of the lactating rat compared with the virgin animal. It was therefore concluded that insulin concentrations are depressed during lactation as a consequence of the pancreas receiving a diminished glycaemic stimulus rather than because of any change in β-cell sensitivity.

Journal of Endocrinology (1990) 125, 81–88
Modulation of growth hormone (GH) secretion and GH mRNA levels by GH-releasing factor, somatostatin and secretagogues in cultured bovine adenohypophysial cells
Authors: Tanner J. W. , Davis S. K. , McArthur N. H. , French J. T. , Welsh T. H.
Published: December 2008

The effects of endogenous hypothalamic neurohormones and activators of second messenger signalling systems on the secretion of GH and on cell content of GH mRNA of cultured bovine adenohypophysial cells were studied. Synthetic bovine GH-releasing factor (bGRF; 100 nmol/l) increased secretion of GH by bovine adenohypophysial cells five-fold relative to control. Forskolin (an adenyl cyclase activator; 10 μmol/l) and the synthetic cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) increased secretion of GH by 1·9- and 1·7-fold respectively, relative to control. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), provided at 1 μmol/l or 10 nmol/l, increased GH secretion by 6·6- and four-fold respectively, relative to control. Somatostatin-14 (SRIF-14) attenuated basal, bGRF-, forskolin- and dbcAMP-stimulated secretion of GH by 40, 49, 47 and 67% respectively, but did not, however, diminish PMA-stimulated GH secretion. The content of GH mRNA in cultured bovine adenohypophysial cells increased 2·2-, 1·7- and 3·2-fold by administration of bGRF, forskolin and PMA respectively, relative to control. Although GH mRNA content was unchanged by SRIF-14 treatment relative to control, SRIF-14 did reduce bGRF-stimulated bGH mRNA content by 67%. This study demonstrates that mechanisms subserving GH secretion in bovine adenohypophysial cells (e.g. adenyl cyclase and protein kinase C) may be coupled with mechanisms which regulate expression of the GH gene or with factors affecting message stability.

Journal of Endocrinology (1990) 125, 109–115
Serum concentrations of thyroid hormones and activity of iodothyronine deiodinase in peripheral tissues of the house musk shrew, Suncus murinus
Authors: Tsukahara F. , Muraki T. , Nomoto T.
Published: December 2008

Serum concentrations of thyroid hormones and the properties of iodothyronine deiodinase in tissues of the house musk shrew, Suncus murinus, were examined and compared with those of the rat. Serum concentrations of thyroxine (T4) and 3,3′,5′-tri-iodothyronine (rT3) were higher, while the serum concentration of 3,5,3′-tri-iodothyronine (T3) was lower in the shrew than in the rat. Among liver, kidney, skeletal muscle, heart, brown adipose tissue (BAT), spleen, lung, testes and thymus homogenates of the shrew, T4 5′-deiodinase (5′D) activity was highest in BAT, and rT3 5′D activity was highest in both the liver and BAT. Intermediate or low T3 5-deiodinase activity was noted in all tissues examined. Activity of 5′D for T4 and rT3 in liver and kidney was much lower, while that of BAT was much higher in the shrew than in the rat. Liver and kidney 5′D may be type-I and that of BAT may be type-II in the shrew, judging from its response to 6-n-propyl-2-thiouracil and iopanoic acid and substrate preference. Thus 5′D of the shrew was similar to that of the rat in type, but was different with respect to its activity in some peripheral tissues. This difference may have relevance to the low T3 state of the shrew.

Journal of Endocrinology (1990) 125, 117–122
The paracrine role of angiotensin in gonadotrophin-releasing hormone-stimulated prolactin release in rats
Authors: Kubota T. , Judd A. M. , MacLeod R. M.
Published: December 2008

It is well known that lactotrophs are in close proximity to gonadotrophs in the lateral region of the pituitary gland, and thus there is interest in interactions between these two types of cell. The present study was undertaken to investigate the role of angiotensin II (AII) in gonadotrophin-releasing hormone (GnRH)-induced prolactin release, and to examine the effect of oestradiol on the paracrine interaction among anterior pituitary cells of young male rats. Over a 3-day period, one group of rats was injected twice with polyoestradiol phosphate (0·5 μg/g body weight; PEP-treated group), and a second with saline (control group). Their anterior pituitary glands were enzymatically dispersed and, subsequently, the cells were allowed to reaggregate for 48 h.

A 20-min perifusion with 100 nmol GnRH/1 increased (P<0·01) prolactin release from these anterior pituitary cell aggregates. The integrated value for prolactin release was 9·1 ±2·9 ng/107 cells. In the PEP-treated group, basal release of prolactin was greater than that in the control group (P<0·01). However, during exposure to GnRH, the integrated amount of prolactin release by the PEP-treated group (12·5 ± 4·8 ng/107 cells) was not significantly different from that of the control group, although in each individual experiment the GnRH-stimulated prolactin release from the PEP-treated cells was higher than that from the cells that had not been exposed to PEP. The release of angiotensin I (AI) from these perifused pituitary aggregates was significantly (P<0·01) increased by GnRH. In contrast, GnRH-stimulated release of prolactin was significantly (P<0·01) suppressed by 100 nmol saralasin/l, a specific AII antagonist, in both control and PEP-treated groups, whereas saralasin did not attenuate GnRH-induced LH release. GnRH-induced LH release was suppressed by PEP treatment during the first 2 min of perifusion, but enhanced throughout the remaining 18 min. In PEP-treated cell aggregates, the release of AI was increased during the later period. These data demonstrate that GnRH is capable of stimulating prolactin release through a mechanism that may involve the release of angiotensin.

Journal of Endocrinology (1990) 125, 225–232
Effect of long-term inhibition of gonadotrophin secretion by the gonadotrophin-releasing hormone agonist, buserelin, on sex steroid secretion and ovarian morphology in polycystic ovary syndrome
Authors: Macleod A. F. , Wheeler M. J. , Gordon P. , Lowy C. , Sönksen P. H. , Conaglen J. V.
Published: December 2008

In order to investigate the effect of long-term suppression of the gonadotrophin axis in polycystic ovary syndrome, eight affected subjects were given s.c. infusions of gonadotrophin-releasing hormone (GnRH) agonist buserelin for 12 weeks. Hormone measurement and ultrasound studies were carried out weekly, from 6 weeks before to 12 weeks after administration of buserelin. An overnight dexamethasone-suppression test was carried out before and after treatment.

Maximal suppression of LH to below the lower limit of that in normal subjects occurred after 6 weeks of treatment with buserelin. Plasma testosterone and androstenedione fell to normal levels during the infusion but reached pretreatment levels during the follow-up period. There was no effect of buserelin on plasma dehydroepiandrosterone sulphate or sex hormone-binding globulin. Ovarian size decreased significantly during the infusion with the disappearance of cysts in six subjects. After cessation of buserelin therapy, there was rapid and spontaneous ovulation which occurred within 3 weeks in all subjects. The results suggest that treatment with this GnRH agonist facilitates ovulation in this condition.

Journal of Endocrinology (1990) 125, 317–325
Effect of angiotensins on electrogenic anion transport in monolayer cultures of rat epididymis
Authors: Wong P. Y. D. , Fu W. O. , Huang S. J. , Law W. K.
Published: December 2008

Confluent monolayers cultured from the rat cauda epididymidis have been shown to respond to angiotensin I (AI) and angiotensin II (AII) when studied under short-circuit conditions and bathed on both sides with Krebs–Henseleit solution. Both the decapeptide AI and the octapeptide AII elicited transient increases in short-circuit current (SCC) when added to the basolateral as well as to the apical surfaces, with the effect of basolateral application greater than that of apical application. The maximal responses produced by AI and AII were similar with median effective concentrations of 20 to 80 nmol/l. The increase in SCC by AII was dependent upon extracellular Cl− and was inhibited by addition of a Cl− channel blocker, diphenylamine 2-carboxylate, to the apical surface. These patterns of activity suggest that the SCC responses to angiotensins result from electrogenic chloride secretion. Pretreating the monolayers with captopril (100 nmol/l), an angiotensin-converting enzyme (ACE) inhibitor, reduced the response to basolateral application of AI, but completely abolished the response to AI added apically. These results suggest that the response to apical addition of AI was due to conversion of AI to AII which interacts with apical angiotensin receptors. This conversion was mediated by ACE which has been detected in epididymal monolayers. Of the endogenous ACE activity, 86% was found to be inhibited by captopril (100 nmol/l).

Responses of the epididymal monolayers to angiotensins were mediated by specific angiotensin receptors. [Sar1,Ile8]-AII, a specific antagonist of the AII receptor, completely inhibited the responses to AI and All but had no effect on the responses to bradykinin and endothelin. The effects of All were mediated by eicosanoid formation since piroxicam, a cyclooxygenase inhibitor, inhibited the AII-induced increase in SCC. This is the first study to demonstrate an effect of angiotensin on epididymal functions. We propose that angiotensin formed locally in the epididymis may play a role in the regulation of electrolyte and fluid transport.

Journal of Endocrinology (1990) 125, 449–456
Purification of human pituitary LH and thyrotrophin by hydrophobic chromatography
Authors: Hiyama J. , Surus A. , Renwick A. G. C.
Published: December 2008

A new preparative procedure is described for the efficient purification of LH and TSH from frozen human pituitary glands. LH and TSH were isolated simultaneously from crude preparations by hydrophobic-interaction chromatography followed by separation and purification by reverse-phase high-performance liquid chromatography in a single step. Highly purified hormones were prepared in good yields (45 mg LH and 20 mg TSH/1000 glands) and with high biological activities; the potencies of purified LH and TSH were 5·8 × NIH-LH-S1 equivalent in an ovarian ascorbic acid depletion assay and 7·1 U/mg against human TSH MRC Research Standard (T1 70/9) in the McKenzie assay respectively. Cross-contamination by other glycoprotein hormones was low.

Journal of Endocrinology (1990) 125, 493–500
OESTROGEN PRODUCTION BY THE GOAT MAMMARY GLAND: TRANSIENT AROMATASE ACTIVITY DURING LATE PREGNANCY
Authors: Peaker M. , Taylor E.
Published: December 2008

Mammary tissue from goats had significantly higher aromatase activity at 148-149 days of pregnancy than earlier in pregnancy or during lactation. Aromatase activity was determined by production of 3H2O from [1 β–3H]androstenedione that was inhibited by 4–hydroxyandrostenedione. The time of maximum aromatization was during peak mammary output of oestradiol–17β described previously in vivo in the same species. Aromatase activity in the mouse mammary gland was low and no peak was observed in late pregnancy. It is concluded that the oestrogens produced by the mammary gland in the late–pregnant goat are formed by aromatization of androgens.
Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor
Authors: Takeda H. , Chodak G. , Mutchnik S. , Nakamoto T. , Chang C.
Published: December 2008

Rat, human, and mouse tissues were stained immunohistochemically using mono- and polyclonal androgen receptor antibodies. Monoclonal antibodies were raised in rats and used to stain human and mouse tissues; polyclonal antibodies were raised in rabbits and used to stain rat tissues. Frozen tissue sections were incubated with the appropriate androgen receptor antibody and staining was completed by the indirect avidin-biotin peroxidase method. A comprehensive survey of rat and mouse tissues was performed. Antibody staining was found exclusively in the nucleus of certain specific cell types, suggesting that the androgen receptor is a nuclear protein.

All male sexual organs in the rat showed strong positive nuclear staining for androgen receptor. Weaker positive reactions were seen in kidney, liver, adrenal cortex and pituitary gland. Furthermore, positive staining for androgen receptor was exhibited in skeletal, cardiac and smooth muscle cells, and central nervous tissue. Female reproductive organs also contained androgen receptor-positive cells. The spleen was found to be the only organ examined which did not stain for androgen receptor. The monoclonal antibody could also demonstrate androgen receptor-positive cells in a human prostatic cancer and in a prostate with benign hyperplasia. These data demonstrate the use of antibodies in revealing cellular/subcellular distribution of androgen receptor in target tissues.

Journal of Endocrinology (1990) 126, 17–25
Sex difference in growth hormone feedback in the rat
Authors: Carlsson L. M. S. , Clark R. G. , Robinson I. C. A. F.
Published: December 2008

Growth hormone inhibits its own secretion in animals and man but the mechanism for this inhibition is unclear: both stimulation of somatostatin release and inhibition of GH-releasing factor (GRF) release have been implicated. We have now studied the GRF responsiveness of conscious male and female rats under conditions of GH feedback induced by constant infusion of exogenous human GH (hGH). Intravenous infusions of hGH (60 μg/h) were maintained for 3 to 6 h whilst serial injections of GRF(1–29)NH2 (0·2–1 μg) were given at 45-min intervals. The GH responses were studied by assaying blood samples withdrawn at frequent intervals using an automatic blood sampling system. We have confirmed that male and female rats differ in their ability to respond to a series of GRF injections; female rats produced consistent GH responses for up to 13 consecutive GRF injections, whereas male rats showed a 3-hourly pattern of intermittent responsiveness. In female rats, multiple injections of GRF continued to elicit uniform GH responses during hGH infusions, whereas hGH infusions in male rats disturbed their intermittent pattern of responsiveness to GRF, and their regular 3-hourly cycle of refractoriness was prolonged. We suggest that this sex difference in GH feedback may be due to GH altering the pattern of endogenous somatostatin release differentially in male and female rats. Such a mechanism of GH autofeedback could be involved in the physiological control of the sexually differentiated pattern of GH secretion in the rat.

Journal of Endocrinology (1990) 126, 27–35
Ontogeny of growth hormone (GH) binding in the domestic turkey: evidence of sexual dimorphism and developmental changes in relationship to plasma GH
Authors: Vasilatos-Younken R. , Gray K. S. , Bacon W. L. , Nestor K. E. , Long D. W. , Rosenberger J. L.
Published: December 2008

The post-hatch ontogeny of hepatic GH binding and its relationship to GH plasma profile characteristics in male and female turkeys of slow- (RBC-2) and fast-growing (F; selected from RBC-2) genetic lines were determined. Specific binding of 125I-labelled recombinant chicken GH to crude hepatic membrane preparations (100 000 g pellet) was determined at 2, 4, 8, 14 and 24 weeks of age for both total (occupied plus free; 4 mol MgCl2/l pretreatment) and free (without MgCl2 pretreatment) binding sites. Characteristics of the plasma GH profile were measured at each age by serial blood sampling through indwelling jugular vein catheters.

When specific binding to either free or total sites was expressed on a whole organ basis (i.e. hepatic GH-binding capacity/bird), binding increased dramatically (P < 0·0001) with increasing age over both lines and sexes. Total binding capacity (free plus occupied sites) per bird was greater for females than for males at 24 weeks of age (P < 0·04), as birds reached sexual maturity, but did not differ between fast- and slow-growing lines at any age. Available binding capacity (free sites) per bird was greater for the faster growing F than RBC-2 line at the older ages when body size was most divergent (14 and 24 weeks of age; P < 0·01, P < 0·06 respectively), but did not differ between sexes.

Correlation analysis at individual ages revealed a progressive change in the nature of the relationship between hepatic GH binding, plasma GH and somatic growth. At 2 weeks of age, when growth velocity of these birds was high relative to later ages, there were no significant correlations among hepatic GH binding, plasma GH and somatic growth. Thereafter, the correlation between total binding capacity and plasma GH concentration progressed from positive (r = 0·34; P < 0·16) to no correlation (r = −0·01; P< 0·97) to increasingly negative, until at the oldest age studied (24 weeks), the correlation was strongly negative (r = −0·55) and significant (P < 0·03). At the older ages, hepatic binding became significantly correlated to measures of somatic growth.

These results indicate that no consistent relationship exists between hepatic GH binding and plasma GH status, over all ages, lines and sexes. Rather different relationships exist at different ages/physiological states. In the growth-divergent lines of turkeys studied here, measures of somatic growth are most strongly correlated with hepatic GH binding at later ages, after the early period of most rapid growth is past, and as mature body size and onset of sexual maturity are approached. The phenomenon of down-regulation of hepatic GH binding as birds approach sexual maturity was suggested by a significant negative correlation between total binding sites and plasma GH at that time.

Journal of Endocrinology (1990) 126, 131–139
Pubertal development and testicular function in the male growth hormone-deficient rat
Authors: Bartlett J. M. S. , Charlton H. M. , Robinson I. C. A. F. , Nieschlag E.
Published: December 2008

The pubertal development of a novel GH-deficient mutant, the dwarf rat, has been evaluated. The establishment of normal spermatogenic function within small testes suggests that GH plays no role in spermatogenic function during puberty and adult life. However, a reduction in testicular size may reflect a reduced Sertoli cell population, suggesting that GH may be of importance in prepubertal testicular development.

Furthermore, marked differences between the homozygous dwarf rat and homozygous GH-deficient mouse mutants (e.g. Snell, Ames, pygmy and little mutants) have been demonstrated. It would appear that the GH deficiency in the rat mutant is far more specific for GH than those hitherto described in the mouse. In contrast to Snell dwarf mice mutants, pituitary and serum concentrations of FSH and LH are normal throughout pubertal development in the dwarf rat. Both spermatogenic function and seminal vesicle function develop normally, whilst in Snell dwarf mice spermatogenic function develops late in life and seminal vesicles remain infantile. Serum and testicular concentrations of androgen are also normal in dwarf rats. Homozygous dwarf rats have been shown to be fertile in previous studies; however, our observations suggest that despite spermatogenesis being qualitatively and quantitatively normal when assessed histologically, reduced testicular size seen in dwarf rats would lead to a reduced daily sperm output in these animals.

The dwarf rat represents a mutant in which the consequences of the selective depletion of GH may be studied on various endocrine systems. The reproductive axis appears to be only partially affected, at an early age, by GH deficiency.

Journal of Endocrinology (1990) 126, 193–201
Development of a new Leydig cell population after the destruction of existing Leydig cells by ethane dimethane sulphonate in rats: an autoradiographic study
Authors: Teerds K. J. , de Rooij D. G. , Rommerts F. F. G. , Wensing C. J. G.
Published: December 2008

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3, or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered.

In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells. The labelling index of the Leydig cells was approximately 100 times higher 21 days after EDS administration than that of the untreated controls, showing that many Leydig cells were formed by proliferation of the newly formed Leydig cells. Thereafter, the labelling index of the Leydig cells gradually decreased, whereas the total number of Leydig cells still increased threefold. At 49 days after EDS administration, the number of Leydig cells was approximately 80% of that in normal adult rats.

It is concluded that the regeneration of Leydig cells after EDS administration is the result of two successive waves of proliferation, namely of the precursor cells (mesenchymal cells) and of the newly formed Leydig cells.

Journal of Endocrinology (1990) 126, 229–236
Transcellular iodide transport and iodination on the apical plasma membrane by monolayer porcine thyroid cells cultured on collagen-coated filters
Authors: Nakamura Y. , Kotani T. , Ohtaki S.
Published: December 2008

Isolated porcine thyroid follicular cells were cultured on a collagen-coated Millipore filter to form a monolayer. The monolayer could translocate 125I added in the medium beneath the filter (basal medium) into the medium above the monolayer (apical medium) and form an iodide concentration gradient of several-fold. Transcellular iodide pump activity was observed when the cells were cultured with TSH in the basal medium. In the absence of TSH, the translocation of iodide was very slow. The concentration of TSH required to activate the iodide pump was 0·1–0·3 mU/ml. Addition of ClO4− to the basal medium inhibited transcellular transport, whilst addition of ClO4− to the apical medium was much less effective.

Constituents labelled with 125I in the apical medium were analysed. The amount of protein-bound 125I measured by acid precipitation was 3–8% of the total radioactivity. The residual radioactivity was found to be iodide ion by paper chromatography. Further analysis by sodium dodecylsulphate–polyacrylamide gel electrophoresis revealed that most of the 125I-labelled protein was at the position of bovine serum albumin which had been added to the culture medium.

The monolayer culture of cells on collagen-coated filter would be a useful experimental system for analysing thyroid cell functions for which the cell polarity is essential.

Journal of Endocrinology (1990) 126, 275–281
Binding of the receptor for 1,25-dihydroxyvitamin D3 to the 5′-flanking region of the bovine parathyroid hormone gene
Authors: Farrow S. M. , Hawa N. S. , Karmali R. , Hewison M. , Walters J. C. , O'Riordan J. L. H.
Published: December 2008

Receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5′-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning − 700 to + 50 bp with 200 μg cytosolic protein gave 288±63% (mean ± s.d.) of binding in the absence of protein. In contrast, there was no significant reaction with the −1350 to − 700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the − 700 to + 50 bp fragment. The receptor-binding site was further defined to −700 to − 100 bp as deletion of the − 100 to + 50 bp did not reduce receptor binding.

Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50 000 Da, which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the −700 to + 50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)2D3 (4 nmol/l) or the presence of KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay.

Autoradiography following incubation of immobilized receptors with the −700 to +50 bp, −485 to −50 bp, −700 to −100 bp and −700 to −485 bp fragments revealed a binding site for the receptor for 1,25-(OH)2D3 between −485 and −100 bp upstream of the initiation site in the bovine PTH gene.

Journal of Endocrinology (1990) 126, 355–359
Comparison of the effects of parathyroid hormone (PTH) and recombinant PTH-related protein on bicarbonate excretion by the isolated perfused rat kidney
Authors: Ellis A. G. , Adam W. R. , Martin T. J.
Published: December 2008

The isolated perfused rat kidney was used to study the effects of amino-terminal fragments of human parathyroid hormone, hPTH(1–34), bovine parathyroid hormone, bPTH(1–84) and of PTH-related proteins, PTHrP(1–34), PTHrP(1–84), PTHrP(1–108) and PTHrP(1–141) on urinary bicarbonate excretion. PTHrP(1–34) (7 nmol/l), bPTH(1–84) (5·5 nmol/l) and hPTH(1–34) (7 nmol/l) had similar effects in increasing bicarbonate excretion with respect to the control. At lower concentrations (0·7 nmol/l) all PTHrP components, but not hPTH(1–34) or bPTH(1–84) increased bicarbonate excretion significantly. Infusions of PTHrP(1–108) and PTHrP(1–141) at 0·7 nmol/l, while associated with a rise in urinary bicarbonate concentration and excretion during the early stages of perfusion, produced a sharp decline in bicarbonate concentration and excretion in the latter part of perfusion. The different peptides produced no significant differences in glomerular filtration rate, fractional excretion of sodium or urine volume. The absence of substantial differences between the effects of hPTH(1–34) and PTHrP(1–34) are as noted in previous studies. The differences between PTHrP(1–108)/PTHrP(1–141) and PTHrP(1–34) demonstrated here are consistent with (1) the clinical manifestations of acidosis in hyperparathyroidism and alkalosis in humoral hypercalcaemia of malignancy, and (2) an independent action of a component of PTHrP beyond amino acids 1–34.

Journal of Endocrinology (1990) 126, 403–408
MOLECULAR EVIDENCE FOR THE PRESENCE OF GROWTH HORMONE RECEPTORS IN THE BOVINE MAMMARY GLAND
Authors: Glimm D.R. , Baracos V.E. , Kennelly J.J.
Published: December 2008

GH receptor messenger RNA (mRNA) was identified and characterized in mammary tissue from normal and GH-treated lactating cows using Northern and in-situ hybridization analyses. One major GH receptor transcript of 4.4 kilobases and a less abundant transcript of 9.2 kilobases were detected in mammary tissue from both normal and GH-treated cows. In-situ hybridization analysis revealed that the GH receptor gene is primarily expressed in the alveolar epithelial cells of mammary tissue. These results are evidence that the lactating mammary gland may synthesize GH receptors. On the basis of these observations it seems likely that the lactating bovine mammary gland is a GH target tissue. This finding challenges the widely accepted view that GH does not directly regulate mammary growth or function.
Influence of pregnancy on G-protein coupling to adenylate cyclase activation in guinea-pig myometrium
Authors: Arkinstall S. J. , Jones C. T.
Published: December 2008

The regulatory factors controlling uterine activity during pregnancy remain unclear in many species. Since myometrial relaxants raise intracellular cyclic AMP, modulation of signalling pathways coupling cell-surface receptors to adenylate cyclase activation could be an important site for control. To assess the functional activity of the stimulatory GTP-binding protein Gs we have measured adenylate cyclase activation by GTP, its non-hydrolysable analogue guanosine 5′-(β-γ-imido)triphosphate (Gpp(NH)p), fluoride, forskolin and manganese in a 50 000 g membrane fraction prepared from the myometrium of non-pregnant, mid-pregnant (30–32 days) and late-pregnant (62–66 days) guinea-pigs (full term 67±2 days). While forskolin- and manganese-dependent enzyme activation was unaltered by pregnancy, maximal stimulation by Gpp(NH)p and fluoride was enhanced by up to 200%. Recovery of adenylate cyclase activity in the 50 000 g fraction was essentially constant at 20–24% of the total activity throughout pregnancy, and thus cannot explain the increases observed. Since guanine nucleotides and fluoride stimulate adenylate cyclase through activating Gs, and forskolin and manganese act at the level of the catalytic unit, these data are consistent with a pregnancy-related increase in Gs functional coupling while adenylate cyclase activity is unaltered. These observations suggest a physiological regulation of myometrial Gs activity during pregnancy which could facilitate hormonal stimulation of adenylate cyclase and contribute to uterine quiescence by increasing uterine sensitivity to relaxants.

Journal of Endocrinology (1990) 127, 15–21