bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence against a role for blastocyst-secreted oxytocin in early pregnancy maintenance in sheep
Authors: Parkinson T. J. , Stewart H. J. , Hunter M. G. , Jones D. S. C. , Wathes D. C. , Flint A. P. F.
Published: December 2008

Analysis of ovine conceptus RNA by slot blotting, Northern analysis and nested polymerase chain reaction failed to detect oxytocin–neurophysin prohormone mRNA. Probes used hybridized with both the 3' end of the prohormone mRNA and the oxytocin-coding sequence. Northern analysis of bovine and porcine conceptus RNA was also negative, and polymerase chain reaction demonstrated oxytocin–neurophysin mRNA in ovine corpus luteum, but not in human corpus luteum or decidua, or in ovine endometrium. Infusion of oxytocin into the uterine lumen in cyclic ewes between days 9 and 19 or 20 after oestrus failed to prolong the luteal phase of the cycle and had no effect on endometrial oxytocin receptor concentrations or uterine prostaglandin F secretion. Oxytocin administered systematically prevented luteolysis and reduced uterine prostaglandin F secretion. Taken together, these data suggest that blastocyst-derived oxytocin is unlikely to contribute to corpus luteum maintenance in early pregnancy. They are inconsistent with a previous report that the ovine blastocyst synthesizes and secretes oxytocin.

Journal of Endocrinology (1991) 130, 443–449
The induction of a specific protease for insulin-like growth factor binding protein-3 in the circulation during severe illness
Authors: Davies S. C. , Wass J. A. H. , Ross R. J. M. , Cotterill A. M. , Buchanan C. R. , Coulson V. J. , Holly J. M. P.
Published: December 2008

The insulin-like growth factors (IGF-I and IGF-II) are almost completely bound in the circulation to specific binding proteins (IGFBPs). These IGFBPs appear to play a pivotal role in maintaining circulating levels and modulating the delivery of the IGFs to the tissues. A large proportion of the circulating IGFs are bound with high affinity to one of the binding proteins, IGFBP-3. The mechanism by which these IGFs are transferred from the circulatory pool to the tissue receptors is at present unclear. Recent studies in late pregnancy have demonstrated the presence of specific proteases which may modify the IGFBPs such that their affinities for the IGFs are reduced. In this paper, we have demonstrated the presence of a heat-sensitive cation-dependent proteolytic enzyme specific for IGFBP-3 in the serum of five severely ill patients. The activity of this protease was found to vary in these patients, becoming more apparent during fasting than when studied after commencement of parenteral nutrition, indicating that one of the influencing factors in the activity of this protease is the nutritional intake of the patient. Age- and sex-matched healthy adults were also studied in a similar protocol, but no proteolytic modification of any of the IGFBPs was found in any of the samples examined. As the levels of both IGF-I and IGF-II were found to be low in the patients, the presence of a circulatory protease suggests that this may be an adaptive response to increase the bioavailability of the IGFs and possibly to improve the nitrogen retention and counter the catabolic state in severe illness.

Journal of Endocrinology (1991) 130,469–473
A LOW MOLECULAR WEIGHT ENDOMETRIAL SECRETORY PROTEIN WHICH IS INCREASED BY OVINE TROPHOBLAST PROTEIN-1 IS A β2-MICROGLOBULIN-LIKE PROTEIN
Authors: Vallet J. L. , Barker P. J. , Lamming G. E. , Skinner N. , Huskisson N. S.
Published: December 2008

Ovine trophoblast protein-1 (oTP-1), stimulates the secretion of several proteins in explant culture of day-12 cyclic ovine endometrium. We partially purified and identified one of these proteins, an 11,000 Mr, pI approx. 6 protein by N-terminal amino acid sequencing and immunoprecipitation using antibody to human β2-microglobulin. The protein was purified from cultures of endometrium collected from day-16 pregnant ewes. The N-terminal amino acid sequence was 40–55% homologous to β2-microglobulin from a variety of species. Antibody to human β2-microglobulin immunoprecipitated the protein and another protein of similar molecular weight but more acidic pi. Using immunoprecipitation of radiolabelled proteins from culture, we demonstrated that oTP-1 increased production of this protein by 40% (P<0.05). We conclude that oTP-1 increases the secretion of a β2-microglobulin-like protein from day-12 non-pregnant endometrium in culture.
Rat thyroid monoamine oxidase (MAO) is regulated by thyrotrophin: evidence that the main form of the enzyme (MAO-A) is not directly involved in iodide organification
Authors: Cabanillas A. M. , Masini-Repiso A. M. , Coleoni A. H.
Published: December 2008

The characteristics and regulation of monoamine oxidase (MAO) were studied in rat thyroid tissue.

A measured Michaelis constant (Km) value of 102 μmol/l was similar to the Km values found in other tissues. Maximal velocity (Vmax) was 1·028 nmol/mg protein per min.

It is known that MAO is present as two isoenzymes, A and B, which are sensitive to clorgyline and deprenyl respectively. The in-vitro effect of graded concentrations of these selective MAO inhibitors was used to estimate the relative proportion of A and B isoenzymes. Clorgyline strongly decreased thyroid MAO activity at concentrations as low as 1 pmol/l while the effect of deprenyl was observed only at concentrations higher than 10 μmol/l. These results indicated that MAO-A is the main form of the enzyme in the rat thyroid.

In-vivo administration of l-thyroxine (5·6–224 nmol/kg) significantly reduced thyroid MAO activity at doses equal to or greater than those which have been reported to inhibit iodine output from the thyroid.

Increased TSH levels, induced either by exogenous TSH or methimazole administration, resulted in a significant increase in thyroid MAO activity. Theophylline, a phosphodiesterase inhibitor and dibutyryl cyclic AMP were also able to stimulate MAO activity when administered in vivo.

Iodide organification (protein-bound 131I) in vivo as well as the relative proportion of the different thyroid iodocompounds were not affected in animals with reduced or increased thyroid MAO activity induced by clorgyline or theophylline respectively.

It was concluded that rat thyroid MAO activity is under the influence of TSH. The adenylcyclase–cyclic AMP system participates in the regulation of enzyme activity as one of the intracellular mediators. An important effect of MAO on thyroid hormone biosynthesis is unlikely since changes in MAO activity did not affect the iodine organification process.

Journal of Endocrinology (1991) 131, 25–31
Growth regulation in the gobiid teleost, Gillichthys mirabilis: roles of growth hormone, hepatic growth hormone receptors and insulin-like growth factor-I
Authors: Gray E. S. , Kelley K. M.
Published: December 2008

Studies of the teleost Gillichthys mirabilis were undertaken to assess the role of GH in regulating hepatic GH receptors, insulin-like growth factor-I (IGF-I) activity and cartilage growth. Hypophysectomized G. mirabilis were injected with saline vehicle or with 10, 100 or 1000 ng tilapia GH (tGH)/g every other day for 2–3 weeks. Growth, judged by increased body weight and length, was inhibited by hypophysectomy and stimulated by increasing tGH doses. Hepatic GH receptors, measured by 125I-labelled tGH binding, were decreased 50% by hypophysectomy. An additional dose-dependent reduction in binding was observed 24 h after tGH injection, but MgCl2 stripping of membranes suggested receptor occupation by exogenous tGH. IGF-I activity, measured by 35SO4 incorporation into oral cartilage explants in vitro, was decreased 50% by hypophysectomy and increased to 200% of intact control levels after injection of 1000 ng tGH/g. In a second experiment, 35SO4 incorporation by oral cartilage from hypophysectomized fish injected with 10, 100 or 1000 ng tGH/g was stimulated to intact control levels. Effects of feeding and tGH injection on in-vitro responsiveness of oral cartilage to recombinant bovine IGF-I (rbIGF-I; 10–1000 ng/ml) were also assessed. Oral cartilage from fed fish showed a parabolic dose–response curve, whereas oral cartilage from starved fish had a lower basal rate of 35SO4 incorporation and a linear dose–response relationship. Oral cartilage from hypophysectomized G. mirabilis showed a significantly attenuated response to rbIGF-I which was restored by tGH injection, suggesting that the GH status of the animal is important for sensitivity of target tissue to IGF-I. Because of its similarity to other vertebrate systems, G. mirabilis presents a good teleost model of growth regulation and of the functions and interactions of GH and IGF-I.

Journal of Endocrinology (1991) 131, 57–66
Effect of thyroid state and cold exposure on rat liver mitochondrial protein mass and function
Authors: Iossa S. , Liverini G. , Barletta A.
Published: December 2008

The effects of thyroid state on liver mitochondrial protein mass was investigated in rats at 24 and 4 °C, as was oxidative phosphorylation using substrates which represent the final catabolic products of the metabolic fuels.

In rats at 24 °C, a significant increase in mitochondrial protein mass (about +40%) was observed only in hyperthyroid animals, while a significant increase due to cold exposure was found in hypothyroid (+ 45%) and euthyroid (+ 35%) rats.

In rats at 24 °C, hypothyroidism significantly decreased the oxidation of glutamate and palmitoyl carnitine but not of pyruvate, while hyperthyroidism only increased the oxidation of palmitoyl carnitine. On the other hand, exposure to cold significantly increased the oxidation of glutamate and pyruvate only in the presence of tri-iodothyronine.

Our results underline not only the fact that a simple and single hypothesis for thyroid effects cannot be adopted, but also that any study concerning oxidative metabolism should be carried out using different substrates and involving different pathways of oxidation.

Journal of Endocrinology (1991) 131, 67–73
Growth hormone regulation of insulin-like growth factor-I mRNA in rat adipose tissue and isolated rat adipocytes
Authors: Vikman K. , Isgaard J. , Edén S.
Published: December 2008

The effects of hypophysectomy and hormonal replacement therapy on insulin-like growth factor-I (IGF-I) mRNA in rat adipose tissue and adipocytes were studied. The effects of GH and IGF-I in vitro on IGF-I mRNA and IGF-I production were also studied in cultured rat adipocytes. Male rats were hypophysectomized at about 50 days of age and given replacement therapy with cortisol (400 μg/kg per day) and thyroxine (10 μg/kg per day). GH was given as a single i.v. or s.c. injection and also as a continuous s.c. infusion for 6 days. Epididymal fat pads were excised and used either for isolation of adipocytes or for determination of IGF-I mRNA in adipose tissue. A solution hybridization assay was used. The IGF-I mRNA content of adipocytes was analysed either immediately after isolation or after short-term (2–3 days) culture with or without GH or IGF-I. Hypophysectomy resulted in a marked decrease in IGF-I mRNA in both tissue and cells. Replacement therapy (in vivo) with cortisol and thyroxine alone had no effect, whereas additional treatment with GH caused a dose-dependent increase in IGF-I mRNA. IGF-I mRNA was also increased after a continuous s.c. infusion of GH. A single i.v. injection of GH (100 μg) resulted in an increase in IGF-I mRNA after approximately 2 h, with maximal levels around 6 h after the injection. In cultured adipocytes, addition of GH to the culture medium increased IGF-I mRNA in a dose-dependent manner and a marked increase was observed with a concentration of GH of 1 ng/ml. Addition of IGF-I (100 ng/ml) had no effect. The increase in IGF-I mRNA after addition of GH (100 ng/ml) was detectable after 3 h. The concentration of IGF-I in the culture medium was increased 24 h after the addition of GH. These results demonstrate that GH induces IGF-I mRNA in both adipose tissue and isolated fully differentiated adipocytes and that this increase in IGF-I mRNA results in increased IGF-I production.

Journal of Endocrinology (1991) 131, 139–145
Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins
Authors: Swennen D. , Rentier-Delrue F. , Auperin B. , Prunet P. , Flik G. , Bonga S. E. Wendelaar , Lion M. , Martial J. A.
Published: December 2008

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/1. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Journal of Endocrinology (1991) 131, 219–227
Expression of hepatic mRNAs for insulin-like growth factors-I and -II during the development of hypothyroid rats
Authors: Gallo G. , de Marchis M. , Voci A. , Fugassa E.
Published: December 2008

The effect of thyroid status on the expression of insulin-like growth factors-I and -II mRNAs in the liver of developing rats has been investigated. Northern blot analyses of the specific mRNA demonstrated the presence of four IGF-II mRNA species which were strongly expressed in fetal liver and progressively declined after birth, becoming undetectable after week 3. This decrease was markedly delayed in the liver of hypothyroid rats. In addition, expression of IGF-I mRNA, absent in fetal liver, began during week 1 after birth and progressively increased with age. This increase was markedly delayed in the liver of hypothyroid rats. The data suggest that thyroid hormones regulate rat development via the co-ordinate expression of hepatic IGF-II and IGF-I mRNAs.

Journal of Endocrinology (1991) 131, 367–372
Absence of pregnancy-induced alterations in tissue insulin sensitivity in the offspring of diabetic rats
Authors: Holemans K. , Aerts L. , Van Assche F. A.
Published: December 2008

We have previously demonstrated insulin resistance in the liver and peripheral tissues of the adult offspring of rats made diabetic with streptozotocin (SDF rats). In this study, a euglycaemic hyperinsulinaemic clamp was used to test the hypothesis that insulin resistance is further aggravated during pregnancy in SDF rats. Normal pregnancy was accompanied by a decrease in the sensitivity of the liver and peripheral tissues to insulin, with a normal responsiveness to insulin. In SDF rats no further decrease in the sensitivity of peripheral tissues to insulin occurred during pregnancy when compared with non-pregnant rats, and the dose–response curves of the glucose metabolic clearance rate during hyperinsulinaemia were similar in pregnant control and pregnant SDF rats. There was, however, a modest decrease in the sensitivity of the liver to insulin during pregnancy in SDF rats.

The normal increase in plasma insulin levels during pregnancy was blunted in SDF rats: this resulted in increased glucose levels in maternal and fetal rats and increased fetal insulin concentrations, features compatible with mild 'gestational diabetes'.

In conclusion, gestational diabetes develops in pregnant SDF rats, although there is no further deterioration in peripheral insulin resistance.

Journal of Endocrinology (1991) 131, 387–393
Rat anterior pituitary neuropeptides following chronic prolactin manipulation: a combined radioimmunoassay and mRNA study
Authors: O'Halloran D. J. , Jones P. M. , Ghatei M. A. , Bloom S. R.
Published: December 2008

Prolactin secretion is highly regulable, and the possibility exists that there are local intrapituitary factors controlling prolactin secretion. Recently, the neuropeptides vasoactive intestinal peptide (VIP), galanin and substance P (SP) have been co-localized to the lactotroph in the female rat. We investigated the effects of alterations in prolactin status in vivo on pituitary and hypothalamic expression of these peptides by specific radioimmunoassays and mRNA analysis. In the anterior pituitary, following haloperidol treatment, the contents of both VIP and galanin were suppressed to below detectable levels. Similarly, after bromocriptine treatment, the content of VIP was decreased to below the detection limit of the assay while galanin (14·2±1·3 vs control 21·0±2·1 fmol/mg, P < 0·05) also showed a significant reduction. The levels of VIP mRNA and galanin mRNA in these groups showed the same qualitative change as their respective peptides. Concurrent treatment with highdose oestrogen modified the VIP peptide response to bromocriptine (1368·7 ±149·2 vs bromocriptine 843·4±82·7 fmol/mg, P<0·05) but not to haloperidol. Oestrogen-induced decreases in galanin content were not influenced by either treatment. The pituitary content of SP showed a fall after oestrogen treatment (1·1±0·01 vs control 6·4±0·8 fmol/mg, P<0·05) which was not significantly altered by either bromocriptine or haloperidol. Likewise, SP mRNA levels in the pituitary were decreased by 90% following oestrogen treatment. Hypothalamic expression of these peptides did not change with any of the treatments. This study demonstrates the tissue and peptide specific influences of prolactin status in regulating anterior pituitary VIP, galanin and SP at the pretranslational level. The results of this study suggest that prolactin status and/or dopaminergic influences are important in the regulation of anterior pituitary neuropeptides.

Journal of Endocrinology (1991) 131, 411–419
Mechanism of secretion of plasma insulin-like growth factor-I into milk of lactating goats
Authors: Prosser C. G. , Fleet I. R. , Davis A. J. , Heap R. B.
Published: December 2008

125I-Labelled insulin-like growth factor-I (IGF-I) was infused as the free form directly into the pudic artery supplying one gland of lactating goats (n = 6). The infusion was for 60 min and 0·4±0·09% (s.e.m.) of the infusate was secreted into milk from the infused gland during its first passage through that gland. A large proportion of the 125I-labelled IGF-I escaped into the systematic circulation and was secreted into milk of both glands. A total of 5·2±0·4% of infused radioactivity was recovered in milk from both glands from 0 to 720 min. Radioactivity consisted of trichloroacetic acid (TCA)-precipitable and -soluble counts which were shown by gel filtration to be authentic IGF-I and degraded products of the peptide. The amount and time course of TCA-soluble radioactivity in milk from both glands was similar, suggesting degradation of 125I-labelled IGF-I at extramammary sites. Maximum specific activity for 125I-labelled IGF-I in milk from the infused gland was reached 80–120 min after the start of infusion and was 2·5-fold greater than milk from the non-infused gland. The time course of appearance of 125I-labelled IGF-I in milk suggests that transfer was via the transcellular pathway and this was further supported by comparing the pattern of transfer of [14C]sucrose and [14C]amino acids. When excess unlabelled IGF-I was included in the infusate, specific activity in milk from the infused gland was reduced to that of the non-infused gland, indicating a competitive and saturable mechanism of secretion for 125I-labelled IGF-I. Comparison of uptake and secretion of 125I-labelled IGF-I into milk from the non-infused gland with that of endogenous immunoreactive IGF-I suggests that vectorial transport of IGF-I across the mammary gland may be a significant contributor of IGF-I levels in milk.

Journal of Endocrinology (1991) 131, 459–466
Most of the circulating insulin-like growth factors-I and -II are present in the 150 kDa complex during human pregnancy
Authors: Gargosky S. E. , Owens P. C. , Walton P. E. , Owens J. A. , Robinson J. S. , Wallace J. C. , Ballard F. J.
Published: December 2008

The aim of this study was to assess the molecular size distribution of insulin-like growth factors (IGFs) complexed to IGF-binding proteins (IGFBPs) in serum of non-pregnant and pregnant women. Sera were fractionated on a size-exclusion column at pH 7·4 to resolve different IGF–IGFBP complexes from unbound IGFs. Each fraction was further chromatographed on a size-exclusion column under acid conditions to dissociate IGFs from binding proteins prior to measurement of the IGF content of the complexes. The IGF-containing fractions from the acid column were assayed specifically for IGF-I and IGF-II. Serum pooled from pregnant women contained more IGF-I and IGF-II than serum from non-pregnant women. In both groups most of the IGF-I and IGF-II was found in a large (150 kDa) complex in serum and the remainder was present in complexes eluting in the 40–50 kDa region at pH 7·4. Some free IGF-I was also detected. Serum fractions collected by size-exclusion chromatography at pH 7·4 were also analysed by Western-ligand blotting to characterize the IGFBPs in the two main IGFBP size classes. In serum pooled from non-pregnant women, the 150 kDa IGF–IGFBP complexes contained a 40–50 kDa IGFBP doublet following Western-ligand blot analysis. This IGFBP co-migrated with a pure IGFBP-3 standard. IGFBPs of 34, 28 and 24 kDa all contributed to the 40–50 kDa IGF–IGFBP complexes of the pH 7·4 chromatograph. In serum pooled from pregnant women, the 40–50 kDa IGFBP-3 doublet and 34 kDa IGFBP were not evident in any fractions from the pH 7·4 column. Thus, although the amounts of IGF-I and IGF-II in 150 kDa IGF–IGFBP complexes were increased during late pregnancy, IGFBP-3 measured by Western-ligand blot analysis of this complex was greatly diminished. The large amount of IGF-I and IGF-II in 150 kDa complexes is strong evidence for the presence of IGFBP-3 in serum during pregnancy, because IGFs and IGFBP-3 are normally present in equimolar amounts in this complex. We suggest that during pregnancy in women, the IGFBP-3 in the 150 kDa complex becomes unstable and this may explain the failure of the Western-ligand blot to detect IGFBP-3.

Journal of Endocrinology (1991) 131, 491–497
ACTIVE IMMUNIZATION WITH A SYNTHETIC PEPTIDE REGION OF GROWTH HORMONE: INCREASED LEAN TISSUE GROWTH
Authors: Pell J.M. , Aston R.
Published: December 2008

The effects of active immunization against a specific peptide region of GH (sequence 134-154) were examined in a species relevant for animal production in vivo. Peptide-immunized lambs exhibited significant increases in carcass protein and water contents and consequently increased carcass weight. The comparatively smaller increases in carcass water and protein contents which occurred in GH-treated lambs were offset by decreased fat accretion. Thus, even though peptide immunization improved lean tissue growth, it did not simply enhance all actions of GH or mimic exogenous hormone administration. This is the first definitive demonstration that endogenously produced antibody may enhance endogenous hormone activity and performance in animals.
ATRIAL NATRIURETIC PEPTIDE IS A PHYSIOLOGICAL INHIBITOR OF ACTH RELEASE: EVIDENCE FROM IMMUNONEUTRALIZATION IN VIVO
Authors: Fink G. , Dow R.C. , Casley D. , Johnston C.I. , Lim A.T. , Copolov D.L. , Bennie J. , Carroll S. , Dick H.
Published: December 2008

The brain is thought to exert a predominantly stimulatory action on ACTH secretion mediated mainly by corticotrophin-releasing factor-41 (CRF-41) and arginine vasopressin (AVP). Several data, however, also point to the existence of an ACTH-inhibiting factor. Atrial natriuretic peptide (ANP), at concentrations found in hypophysial portal blood, inhibits ACTH release in vitro. The aim of the present studies was to use ANP immunoneutralization to determine whether ANP does in fact inhibit ACTH release in vivo. Intracerebroventricular infusion (I μl/min for 30 min) of sheep anti-ANP serum into male rats anaesthetized with sodium pentobarbitone had no significant effect on jugular venous plasma concentrations of ACTH or LH but did decrease significantly the plasma concentrations of prolactin. Intravenous infusion of 0.8 ml sheep anti-ANP serum but not control (non-immune) sheep serum, through an indwelling intra-atrial cannula in conscious male rats resulted in a marked and significant increase in plasma ACTH and corticosterone concentrations. The ACTH and corticosterone response to a 30-s ether stress was not significantly potentiated in the same conscious rats infused with anti-ANP serum. Intra-atrial infusion of anti-ANP did not significantly affect plasma prolactin, LH, glucose or sodium concentrations or plasma osmolality. These results show for the first time that ANP is a potent inhibitor of ACTH secretion in the conscious male rat and that, therefore, ANP is a hypothalamic neurohormone which is likely to play an important inhibitory role in the neural control of ACTH release.
Gonadal steroids effect similar regulation of gonadotrophin subunit mRNA expression in both male and female rats
Authors: Dalkin A. C. , Paul S. J. , Haisenleder D. J. , Ortolano G. A. , Yasin M. , Marshall J. C.
Published: December 2008

Gonadal steroids can act both indirectly via gonadotrophin-releasing hormone (GnRH) and directly on the pituitary to regulate gonadotrophin subunit gene expression. Recent studies to assess a possible direct action at the pituitary have shown that testosterone, when given to males in the absence of endogenous GnRH action, selectively increases FSH-β mRNA concentrations. Conversely, in females, oestradiol appears to regulate gonadotrophin subunit mRNAs primarily via GnRH. The present study was designed to determine whether these differing results reflect specific actions of the gonadal steroids themselves or different responses of the pituitary gonadotroph cells in males and females.

Rats which had been castrated 7 days earlier were given silicone elastomer implants (s.c.) containing oestradiol (plasma oestradiol 68 ± 4 ng/l) in males or testosterone (plasma testosterone 3·5 ± 0·3 μg/l) in females in the absence or presence of a GnRH antagonist. Seven days later pituitaries were removed and steady-state mRNA concentrations measured by dotblot hybridization. In males, oestradiol reduced LH-β and FSH-β but not α mRNA. The antagonist reduced levels of all three subunit mRNAs in males and the addition of oestradiol had no further effect, suggesting that oestradiol regulates gonadotrophin subunit gene expression in males by suppressing GnRH secretion. In females, testosterone reduced all three subunit mRNAs though FSH-β remained threefold higher than in intact animals. The GnRH antagonist was as effective as testosterone alone and reduced α and LH-β to levels found in intact animals. FSH-β mRNA was partially reduced by antagonist alone in ovariectomized females but the addition of testosterone increased FSH-β twofold versus antagonist alone (as has been observed in males). These findings, together with earlier data, suggest that testosterone increased FSH-β twofold versus antagonist alone (as has been observed in males). These findings, together with earlier data, suggest that testosterone reduces gonadotrophin subunit mRNAs by inhibiting GnRH secretion and also acts directly on the gonadotroph to increase steady-state FSH-β mRNA concentrations in both males and females.

Journal of Endocrinology (1992) 132, 39–45
Release of corticotrophin-releasing factor-41, arginine vasopressin and oxytocin from rat fetal hypothalamic cells in culture: response to activation of intracellular second messengers and to corticosteroids
Authors: Hu S.-B. , Tannahill L. A. , Biswas S. , Lightman S. L.
Published: December 2008

The effects of the activation of protein kinase A (PKA), protein kinase C (PKC) and corticosteroids were investigated on the release of corticotrophinreleasing factor-41 (CRF), arginine vasopressin (AVP) and oxytocin from rat fetal hypothalamic cells in culture. Both forskolin and PMA (phorbol 12-myristate 13-acetate) increased CRF, AVP and oxytocin release, while dexamethasone and aldosterone only reduced basal secretion of CRF. Both steroids also inhibited forskolin-induced CRF, AVP and oxytocin responses to PMA. These data provide direct evidence for a role for both PKC- and PKA-mediated mechanisms in the regulation of CRF, AVP and oxytocin release and for differential effects of both glucocorticoids and mineralocorticoids on PKA- and PKC-stimulated responses.

Journal of Endocrinology (1992) 132, 57–65
Expression of an abundant α-class glutathione S-transferase in bovine and human adrenal cortex tissues
Authors: Meikle I. , Hayes J. D. , Walker S. W.
Published: December 2008

Bovine adrenal cortex tissue expresses high levels of glutathione S-transferase (GST) from each of the α, μ and π gene families. We describe the purification and characterization of an abundant α-class GST from this tissue that has not been identified previously because of its failure to bind to S-hexylglutathione–Sepharose 6B (S-hexG-Ag). This enzyme has been affinity purified on glutathione–Sepharose 6B (GSH-Ag) and was obtained in a highly purified form by employing S-hexG-Ag to remove the bulk of GST before chromatography on GSH-Ag. The purified GST eluted from GSH-Ag was found to exhibit marked peroxidase and Δ5-ketosteroid isomerase activities (19·2 and 1·67 U/mg respectively). The bovine enzyme also showed high GST activity towards 4-hydroxynonenal (5·09 U/mg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the bovine GST contains two distinct polypeptides, one with an Mr of 25 900 and the other with an Mr of 26 500.

An abundant α-class GST was also purified from human adrenal cortex that possessed properties which were similar to the bovine α-class GST described above; however, unlike the bovine enzyme, the corresponding human α-class GST bound to S-hexG-Ag. As with the bovine enzyme, the purified human GST displayed marked peroxidase and isomerase activities (27 and 4·02 U/mg respectively). Further analysis on SDS-PAGE (Mr 25 800) and reverse-phase highperformance liquid chromatography established that this abundant α-class GST in human adrenal cortex is equivalent to the human liver GST B1B1 enzyme.

As both human and bovine adrenal cortex contain high levels of α-class GST with similar catalytic properties, we discuss the possible functions of these enzymes in this tissue.

Journal of Endocrinology (1992) 132, 83–92
Pituitary adenylate cyclase-activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3
Authors: Propato-Mussafiri R. , Kanse S. M. , Ghatei M. A. , Bloom S. R.
Published: December 2008

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH2(1–38) (PACAP-38) and PACAP-NH2(1–27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH3, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH3 and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH3 cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/1).

Journal of Endocrinology (1992) 132, 107–113
A biphasic effect of noradrenaline on renin release from rat juxtaglomerular cells in vitro is mediated by α1- and β-adrenoceptors
Authors: Takagi M. , Atarashi K. , Matsuoka H. , Sugimoto T.
Published: December 2008

The direct effect of noradrenaline on renin release from juxtaglomerular (JG) cells in vitro were investigated in a dynamic superfusion system of dispersed rat renal cortical cells. At low concentrations (1–100 nmol/l), noradrenaline stimulated renin release in a dose-dependent manner, while at higher concentrations (0·1–1 mmol/l) it inhibited renin release. The stimulatory effect of 0·1 μmol noradrenaline/l was completely blocked by a β-adrenoceptor antagonist, propranolol (0·1 μmol/l). When applied at concentrations of 1 μmol/l or 10 μmol/l, noradrenaline had no consistent effect on renin release, although 10 μmol noradrenaline/l had an inhibitory effect in the presence of propranolol (0·1 μmol/l). The inhibitory effect of noradrenaline (0·1 mmol/l) was converted to a stimulatory effect by the addition of an α1-adrenoceptor antagonist (bunazosin, 1 μmol/l), but was not altered by the addition of an α2-adrenoceptor antagonist (yohimbine, 1 μmol/l). These results indicate that low concentrations of noradrenaline directly stimulate renin release from JG cells by the activation of β-adrenoceptors, while high concentrations of nor-adrenaline inhibit renin release by the activation of α1-adrenoceptors. Accordingly, a dynamic balance may exist between β-adrenergic stimulation and α1-adrenergic depression of renin release.

Journal of Endocrinology (1992) 132, 133–140
Effect of dietary protein intake and intravenous glucose infusion on plasma concentrations of insulin-like growth factor-I in lambs
Authors: Kriel G. V. , Bryant M. J. , Lomax M. A.
Published: December 2008

Two separate experiments were carried out to examine the effect of dietary protein intake on basal and GHstimulated plasma insulin-like growth factor-I (IGF-I) concentrations during either saline or glucose infusion into the jugular vein. In experiment 1, six castrated male lambs (27·1 ± 1·2 kg live weight (LW)) were fed a diet restricted in both metabolizable energy (ME; 0·18 MJ/kg LW per day) and nitrogen (2·0 g/kg LW per day) intakes, while in experiment 2 a further six lambs were fed a similar restricted ME intake but an increased nitrogen intake (3·0 g/kg LW per day). In both experiments glucose (experiment 1, 0·009 mmol/kg LW per min; experiment 2, 0·015 mmol/kg LW per min) or saline (0·25 ml/min) was infused for 6 days and plasma samples were obtained from the jugular vein at hourly intervals on day 4 (basal) or on days 5 and 6 after an i.v. GH challenge. In experiment 1 there was no increase in plasma IGF-I concentrations in response to the GH challenge during saline infusion, but during glucose infusion the plasma concentration of IGF-I increased to a peak after 24 h and declined over the next 20 h. Basal concentrations of IGF-I, insulin and glucose were significantly higher during glucose infusion. In experiment 2 the area under the IGF-I peak in response to the GH challenge was the same for the infusions of saline and glucose but the peak value for IGF-I was significantly higher during glucose infusion due to higher concentrations in the basal period. It is concluded that feeding a dietary intake of metabolisable energy and nitrogen which is only just sufficient to meet the requirements for maintenance of body tissues completely abolishes the increase in plasma IGF-I in response to GH challenge. Increasing the nitrogen supply in the diet or i.v. infusion of glucose re-establishes the IGF-I response to GH challenge as well as increasing basal IGF-I concentrations. The potential involvement of circulating concentrations of insulin, glucose and amino acids in the control of plasma IGF-I levels are discussed.

Journal of Endocrinology (1992) 132, 195–199
Hormone production in vivo and in vitro from follicles at different stages of the oestrous cycle in the sheep
Authors: Mann G. E. , McNeilly A. S. , Baird D. T.
Published: December 2008

This experiment was undertaken in order to investigate the production of inhibin, oestradiol and androstenedione by ovarian follicles at different stages of the oestrous cycle in sheep. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24–30, 36 and 60 h after the induction of luteal regression by an injection of 125 μg cloprostenol on day 10 of the luteal phase. Samples of jugular and ovarian venous blood were collected under anaesthesia and ovaries were then removed and all follicles larger than 3 mm diameter dissected out and incubated in medium for 2 h.

After injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion rate of progesterone. The ovarian secretion rate of inhibin was similar at all stages of the follicular phase and during the luteal phase while, in contrast, the secretion rate of oestradiol was significantly (P < 0·05) elevated in the group 24 h after injection of cloprostenol. There was good correlation between the in-vivo ovarian secretion rate and production rate during incubation in vitro for both inhibin (r = 0·57) and oestradiol (r = 0·60). When follicle diameter was compared with in-vitro hormone production there was good correlation for inhibin (r = 0·72) with larger follicles producing more inhibin, while the value for oestradiol was somewhat lower (r = 0·57) owing to the presence of large atretic follicles with low oestradiol production. Androstenedione production showed a lower correlation with follicle diameter (r = 0·39). When the four time periods were compared separately, there were significantly (P < 0·05) more follicles with high in-vitro oestradiol production (> 90 fmol/min) in the group at 36 h than in the other three groups, while inhibin release in relation to follicle size was similar in the four groups. Large oestrogenic follicles were responsible for 90% of the total oestradiol production during culture while only providing 55% of the total inhibin production, with large non-oestrogenic and small follicles contributing 33% and 12% of inhibin production respectively.

From the results of this study we conclude that while oestradiol is mainly produced by the large oestrogenic follicles, a considerable amount of inhibin is also produced by large non-oestrogenic and small follicles. We also found that a lack of variation in inhibin secretion rate in the intact animal was paralleled by a lack of variation in the pattern of inhibin produced from individual follicles.

Journal of Endocrinology (1992) 132, 225–234
The role of sex steroids in the formation of sex-differentiated concentrations of corticosteroid-binding globulin in rats
Authors: Mataradze G. D. , Kurabekova R. M. , Rozen V. B.
Published: December 2008

The role of sex steroids in the programming of the level of serum corticosteroid-binding globulin (CBG) in the rat has been studied at different stages of ontogenesis.

The CBG content in the serum of mature female rats was 2·5 times higher than that in male rats. Sexual dimorphism of CBG content was absent in immature animals of 3–4 weeks of age. Castration of mature rats led to a 40–50% increase in CBG content. The CBG concentration in mature females or castrated adult males treated with testosterone propionate (TP; 3 mg/day for 4 days) was decreased by 40–50% compared with vehicle-treated rats. Oestradiol injection (1 μg/day for 4 days) had no influence on CBG levels in mature male and ovariectomized adult female rats.

Immature rats were castrated on days 1, 7, 14, 21, 28 or 35 of age and the CBG level was determined at 10–12 weeks of age. The CBG content of rats castrated up to day 28 of age was 2·5 times higher than that in mature males and did not differ from that in mature females. The CBG content of male rats castrated on day 35 of age was the same as that of adult castrated males. The CBG level in castrated rats treated with TP (1·25 mg for days 1–3 or 300 μg/day for 5 days after castration at day 7 up to day 26) did not differ from that in controls (i.e. vehicle-treated rats). TP injection into castrated rats on days 29–33 of age (300 μg/day) led to a 40–50% decrease in CBG level when compared with controls.

Ovariectomy of rats at different ages (on days 1 or 28 or 3 months) did not affect CBG concentration. TP injection (300 μg/day) into ovariectomized rats on days 29–33 had the same effect on CBG concentration as in males.

Gonadectomized rats treated with diethylstilboestrol on days 29–33 (100 μg/day) had the same CBG concentrations as TP-treated rats.

It was concluded that there is a short period during ontogenesis, from days 29 to 35 of age, which is critical for irreversible masculinization of the CBG concentration in male rats.

Journal of Endocrinology (1992) 132, 235–240
Acute effects of oestradiol and progesterone on melittin- and gonadotrophin-releasing hormone-induced LH secretion
Authors: Ortmann O. , Johannsen K. , Knuppen R. , Emons G.
Published: December 2008

It is well established that oestradiol and progesterone modulate gonadotrophin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells. Short-term oestradiol and long-term progesterone treatment exert inhibition, while short-term progesterone and long-term oestradiol treatment lead to enhancement of GnRH-stimulated LH secretion. There are several lines of evidence to suggest that the steroid effects might be mediated via a mechanism involving modulation of the GnRH signal-transduction system. To evaluate the role of arachidonic acid, which serves as an intracellular signal transducer by itself or its lipoxygenase metabolites, in the mediation of oestradiol and progesterone actions, we examined their effects on melittin (activator of phospholipase A2)-stimulated LH secretion. When pituitary cells from adult female rats were treated for 48 h with 1 nmol oestradiol/l or 1 nmol oestradiol/l plus 100 nmol progesterone/l, GnRH (1 nmol/l)-induced LH secretion was stimulated or inhibited respectively. However, melittin (10–300 nmol/l)-stimulated LH secretion remained unaffected after such treatment. Short-term treatment with oestradiol inhibited GnRH-induced LH secretion while progesterone treatment of oestradiol-primed cells led to a stimulatory effect. Interestingly, melittin-stimulated LH secretion was influenced in the same way after the short treatment paradigm. Perifusion studies were performed to assess the kinetics of these acute steroid actions further. Four separate perifusion chambers were continuously perifused with medium and stimulated for 2 min with 1 nmol GnRH/l or 1 μmol melittin/l every 50 min in a pulsatile fashion. When 1 nmol oestradiol/l was added to the perifusion medium after the application of an initial control pulse, GnRH- and melittin-stimulated LH secretion were inhibited by 69 and 61% respectively. This effect was present after 50 min. When oestradiol-primed cells were treated with 100 nmol progesterone/l starting after the initial GnRH or melittin pulse, an acute stimulatory effect was observed in response to both stimuli after 50 min. LH release was enhanced by up to 279 (GnRH) or 419% (melittin) compared with the control pulse. The kinetics of inhibited or stimulated pulsatile LH secretion were virtually identical when GnRH or melittin were used as stimuli. These results demonstrate that short-term oestradiol or progesterone treatment modulate arachidonic acid-mediated LH secretion in a similar fashion to GnRH-induced LH secretion, while long-term oestradiol or progesterone treatment only affected GnRH-induced LH secretion.

Journal of Endocrinology (1992) 132, 251–259
Characterization of chicken lung angiotensin I-converting enzyme
Authors: Polanco M. J. , Miguel J. L. , Agapito M. T. , Recio J. M.
Published: December 2008

Angiotensin I(AI)-converting enzyme (ACE) (EC 3.4.15.1) was solubilized from the membrane fraction of chicken lung using trypsin and nonidet P40 extraction, and then purified to homogeneity by captopril affinity chromatography. Comparison of trypsin-extracted and detergent-solubilized membrane-bound converting enzyme by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane-binding sequence contributed to a large extent to the size and charge of the enzyme. Both forms of the enzyme were glycoproteins but they differed in the glucidic content; 4·5% by weight of the enzyme in the trypsin-extracted ACE and 15% by weight of the enzyme in the detergent-solubilized ACE. In both cases hexoses were the most abundant residues. Both forms of the enzyme were found to contain 1 g-atom zinc/mol enzyme.

The purified enzymes did not only split Hip-His-Leu but also AI and bradykinin. The Michaelis constant (Km) and maximum velocity (Vmax) values of the trypsin-extracted ACE for Hip-His-Leu were 52 × 10−5 mol/l and 15·36 nmol/min respectively, and for AI they were 7·8 × 10−5 mol/l and 0·45 nmol/min respectively. The Km and Vmax values of the detergent-solubilized ACE for Hip-His-Leu were 32 × 10−5 mol/l and 11·75 nmol/min respectively, and for AI they were 6·5 × 10−5 mol/l and 0·97 nmol/min. Both forms of the enzyme required the same chloride ion concentration (0·3 mol/l) for the optimum enzyme activity and showed the same pH optimum (8·5) with Hip-His-Leu as substrate. Both were inhibited by EDTA in a similar manner. In addition, they were inhibited by the peptides AII and His-Leu, but captopril was the most potent inhibitor with inhibition constant values of 1·6 nmol/l and 2·0 nmol/l for the trypsin-extracted and the detergent-solubilized ACE respectively.

Journal of Endocrinology (1992) 132, 261–268
Effect of increased production of growth hormone on body composition in mice: transgenic versus control
Authors: Searle T. W. , Murray J. D. , Baker P. J.
Published: December 2008

The body composition (water, fat, protein and ash) of male and female transgenic mice which had a sheep metallothionein 1a–sheep growth hormone fusion gene and their non-transgenic controls was determined at intervals from birth to 21 days of age (weaning) in 66 mice of each group, and in an additional 64 mice over the period 25 to 98 days of age. Overall 520 mice were analysed. Weaned mice were starved over-night prior to slaughter. Food was available ad libitum. and, after weaning, a zinc sulphate supplement was added to the drinking water to initiate expression of the transgene.

Growth and body composition were similar in all groups before weaning. From 39 days of age, transgenic females became progressively heavier than corresponding controls, being 60% heavier at the end of the experiment. They contained less fat, more water and slightly less ash than did controls of the same live weight but similar amounts of protein. When examined on a fat-free basis, they had less protein and ash and more water than corresponding controls. Appropriate linear and quadratic regression equations are presented to describe the above relationships.

Growth and body composition were more variable in transgenic males but, on average, similar to controls.

Journal of Endocrinology (1992) 132, 285–291