bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical and biological characterization of chicken insulin-like growth factor-II
Authors: Kallincos N. C. , Wallace J. C. , Francis G. L. , Ballard F. J.
Published: December 2008

Chicken insulin-like growth factor-II (cIGF-II) has been characterized by amino acid sequencing, by its receptor and binding protein interactions and by its biological activities in cultured cells in order to help define the significance of the peptide in the growth process. Chicken IGF-II has an N-terminal region and several amino acid substitutions in the mid-peptide region that differ from the mammalian growth factor. Nevertheless, cIGF-II was indistinguishable from ovine IGF-II in all assay systems, including those involving chicken receptors and chicken binding proteins. Thus the amino acid substitutions did not modify the biological activities. In chick embryo fibroblasts, labelled bovine IGF-II or cIGF-II bound to a receptor with size and specificity properties expected for a type 1 IGF receptor, except that IGF-I competition for binding was less than IGF-II competition. No evidence for a type 2 receptor was obtained. The relatively lower biological activity of IGF-I compared with IGF-II in chick embryo fibroblasts contrasts with the much higher potency of IGF-I in rat L6 myoblasts. This difference can be explained by a combination of an inhibitory, IGF-II-specific binding protein produced only by the rat cells as well as the unusual receptor specificity of the chicken cells.

Journal of Endocrinology (1990) 124, 89–97
3,5,3′-Tri-iodothyronine enhances sugar transport in rat thymocytes by increasing the intrinsic activity of the plasma membrane sugar transporter
Authors: Segal J. , Ingbar S. H.
Published: December 2008

We have shown that 3,5,3′-tri-iodothyronine (T3) produces a prompt increase in sugar transport in rat thymocytes by increasing the maximal velocity without changing the Michaelis–Menten constant of the plasma membrane sugar transport system. To elucidate further the mechanism of this effect, we have now assessed the influence of T3 on the number and affinity of sugar transporters in thymocytes, measured as the sugar (2-deoxyglucose; dGlc)-displaceable binding of cytochalasin B. Cytochalasin B inhibited in a dose-related manner the uptake of dGlc by rat thymocytes with inhibition constant values of 0·19 and 0·22 μmol/l in the presence and absence of T3 respectively. Binding of cytochalasin B by the sugar-displaceable sites was rapid and saturable, demonstrating a single class of sites having an apparent dissociation constant of 0·33 ± 0·02 (s.d.) μmol/l and maximal binding capacity of 3·73±0·48 pmol/20 × 106 cells (11·2±1·4 × 104 sites/thymocyte). In the rat thymocyte, sugar transporters were found to be located in two major subcellular pools, the plasma membrane and microsomes, the latter being about twice the size of the former. In these subcellular compartments, as well as in the intact cell, binding of [3H]cytochalasin B by the sugar-displaceable sites constituted about 40% of total cytochalasin B binding.

3,5,3′-Tri-iodothyronine in concentrations that stimulated uptake of dGlc by thymocytes had no effect on [3H]cytochalasin B binding (total and sugar-displaceable) in the intact cell and in the plasma membrane and microsomal compartments, nor did it influence the affinity and number of sugar transporters. From these observations we conclude that, in the rat thymocyte, T3 acts to increase sugar uptake by increasing the intrinsic activity, i.e. the efficiency, rather than the number of sugar transporters.

Journal of Endocrinology (1990) 124, 133–140
Inhibitory effect of the uterus on plasma and pituitary FSH in rats
Authors: Biro J. C. , Eneroth P.
Published: December 2008

Plasma concentrations of FSH and LH were measured in ovariectomized, ovohysterectomized, hysterectomized and sham-operated adult, non-pregnant rats at 3, 14, 21 and 28 days after operation. From day 21 after the operation onwards, there were higher concentrations of FSH in plasma in ovohysterectomized than in ovariectomized animals. The concentration of LH was not influenced by hysterectomy. The inhibitory response of FSH and LH to a single dose of oestradiol was not altered by any of the operations. By 2 weeks after surgery, pituitary FSH content had increased in ovohysterectomized animals compared with ovariectomized ones, but this difference was eliminated when ovohysterectomized animals were treated with crude uterine extract. Pituitary contents of LH and prolactin were not influenced by hysterectomy or by treatment with uterine extract, thus indicating the specificity of an inhibitory effect of the uterus on FSH levels. Treatment of hysterectomized and intact animals with uterine extract resulted in a reduction in the weight of the ovaries of 23–38% (P<0·05), indirectly showing the presence of an FSH-inhibiting substance in the extract.

Fractionated uterine extract inhibited FSH synthesis by rat pituitary cells in vitro, but had no effect on LH synthesis. Chromatographic analysis indicated that the FSH-inhibiting substance in the uterus has a molecular weight of 10 000–20 000.

Journal of Endocrinology (1990) 124, 183–189
The plasma pattern of growth hormone in conscious rats during late pregnancy
Authors: Carlsson L. , Edén S. , Jansson J.-O.
Published: December 2008

The plasma GH levels of female rats during late pregnancy were determined using an automatic method for repetitive blood sampling from conscious animals. The plasma GH patterns were analysed by a pulse analysis computer program (PULSAR). The mean plasma GH levels were about twofold higher in pregnant females on days 15, 18 and 22 of gestation than in age-matched non-pregnant females. The basal plasma GH levels were also increased, while there was no change in GH pulse amplitude or frequency. The augmentation of GH release was even more pronounced on day 20 of gestation, with a fourfold increase in mean plasma GH levels compared with those in non-pregnant females. This increase reflected an increase in both basal plasma GH levels and GH pulse amplitude, but there was no increase in pulse frequency. In female rats that delivered on day 22 of gestation, the basal and mean plasma GH levels increased during parturition.

Pregnant females consistently responded to multiple i.v. infusions of 1 μg human GH-releasing factor analogue (hGRF(1–29)-NH2) given at 45-min intervals on day 18 of gestation. Both basal and GRF analogue-stimulated plasma GH levels were undetectable after hypophysectomy of pregnant rats.

The present study demonstrates an increase in basal plasma GH levels during late pregnancy and a marked increase in both basal plasma GH levels and GH pulse amplitude on day 20 of gestation. Furthermore, hypophysectomy of pregnant rats results in undetectable GH levels, indicating that the high levels of GH during pregnancy are derived from the pituitary.

Journal of Endocrinology (1990) 124, 191–198
Growth hormone (GH) autofeedback action in the neonatal rat: involvement of GH-releasing hormone and somatostatin
Authors: Cella S. G. , De Gennaro Colonna V. , Locatelli V. , Moiraghi V. , Loche S. , Wehrenberg W. B. , Müller E. E.
Published: December 2008

It is known that in adult rats, GH by itself and by promoting secretion of the somatomedins acts at the level of the hypothalamus to trigger release of somatostatin and decrease output of GH-releasing hormone (GHRH), thereby inhibiting further secretion of GH. To assess whether these mechanisms are already operative in the early postnatal period, we have evaluated the effect of short-term administration of GH in 10-day-old rats. Twice-daily s.c. administration of 25 μg human GH/rat, from days 5 to 9 of life, significantly reduced pituitary content of GH, decreased hypothalamic levels of GHRH mRNA and abolished the in-vivo GH response to a challenge dose of GHRH (20 ng/100 g body weight, s.c.). GHRH (20 ng/100 g body weight, twice daily, s.c.) given concomitantly with the GH treatment, completely counteracted the inhibitory effect of the latter on pituitary content of GH and restored to normal the in-vivo GH response to the GHRH challenge. These data indicate that impaired secretion of GHRH is involved in the inhibitory effect elicited by GH treatment in infant rats. However, concomitant involvement of hypothalamic somatostatin as a result of GH treatment cannot be ruled out. In fact, pituitaries from rats pretreated with GH responded in the same manner as pituitaries from control rats to the GHRH challenge in vitro.

Journal of Endocrinology (1990) 124, 199–205
Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C
Authors: Hirst J. J. , Rice G. E. , Jenkin G. , Thorburn G. D.
Published: December 2008

The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4).

These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum.

Journal of Endocrinology (1990) 124, 225–232
Pharmacological effects of the continuous administration of an LHRH agonist in the ageing male rat: comparison with orchidectomy
Authors: Amir-Zaltsman Y. , Ausher Y. , Gayer B. , Lichter S. , Serour F. , Birkenfeld S. , Kohen F.
Published: December 2008

The age-related changes in tissue response to chronic treatment for 1 month with a potent LHRH agonist were investigated in the ageing male rat, and the observed pharmacological effects were compared with orchidectomy. In both young (4 months) and old (22 months) rats, treatment resulted in a significant decrease in the weights of prostates and testes, a decrease in plasma LH and testosterone levels, a loss of LH receptors in the testes and in a complete depletion of prostatic nuclear androgen receptors, reaching levels observed after castration. In young rats, treatment with an LHRH agonist or orchidectomy induced a three- or sixfold increase in prostatic creatine kinase (CK) activity which may have been induced by the local stimulatory effect of oestradiol arising from the conversion of precursor steroids secreted by the adrenal. On the other hand, in old rats, 7 days after orchidectomy or after treatment with an LHRH agonist a twofold increase or no change was induced in prostatic CK activity respectively. SDS gel electrophoresis patterns of cytosolic prostatic proteins of young rats treated with an LHRH agonist or young rats orchidectomized 7 days previously revealed the presence of several intensified proteins, two of them having apparent molecular weight of 67 kDa and 43 kDa, whereas in the old rats treated with LHRH agonist or old rats castrated 7 days previously, these two proteins were not intensified. The results of this study confirmed that continuous treatment with an LHRH agonist to young and old rats induces medical castration since the pharmacological effects observed were the same as those induced with surgical castration. However, in the old rats, the lack of an increase in prostatic CK activity upon treatment with LHRH agonist, and the moderate increase in CK activity upon orchidectomy, suggest that prostatic cells in older rats have decreased sensitivity to hormonal manipulation.

Journal of Endocrinology (1990) 124, 261–268
Characterization of growth factor activity produced by fetal mouse osteoblasts
Authors: Slootweg M. C. , van Buul-Offers S. C. , Hoogerbrugge C. M. , Herrmann-Erlee M. P. M. , van den Eijnden-van Raaij A. J. M. , Duursma S. A. , van Zoelen E. J. J.
Published: December 2008

Bone matrix contains a variety of growth factors, but little is known of osteoblastic production of such materials. The present study assesses growth factor activity, chromatographed on acidic Bio-Gel P-100, secreted into conditioned media of primary cultures of fetal mouse calvaria. The cultures produced insulin-like growth factor-I (IGF-I), determined by radioimmuno-assay of molecular weights 20 and 10 kDa. IGF-II, determined by radioreceptor assay, was present at 20–29 and 7 kDa. The IGF peaks at 20, 10 and 7 kDa were all mitogenic in MCF-7 cells. Proteins of several different molecular weights were also present that specifically bound IGF-I and IGF-II. Transforming growth factor-β (TGF-β), assayed in a system for inhibition of growth, was also produced. Both activated and latent forms were present, and part of the TGF-β was TGF-β2. The absence of mitogenic activity in the bmolecular range of platelet-derived growth factor, assayed in 3T3 fibroblasts, makes it unlikely that mouse osteoblasts produce this growth factor.

Journal of Endocrinology (1990) 124, 301–309
Plasma clearance of chicken and human insulin-like growth factor-I and their association with circulating binding proteins in chickens
Authors: Francis G. L. , McMurtry J. P. , Johnson R. J. , Ballard F. J.
Published: December 2008

We have investigated the clearance of 125I-labelled chicken and recombinant human insulin-like growth factor-I (IGF-I) from the circulation of chickens as well as the role that IGF-binding proteins play in this process. Analysis of plasma samples by high-performance liquid chromatography (HPLC) neutral gel permeation on a TSK G3000SW column indicated that the i.v. injected radioactivity was rapidly partitioned between at least three pools. Most of the radioactivity occurred in a complex with binding protein(s), while smaller amounts of radioactivity chromatographed in the free IGF-I peak or appeared as low molecular weight degradation products. The labelled chicken and human IGF-I were rapidly cleared during the first 90 min. The calculated half-life for total labelled IGF-I during this period was 54 min for the chicken tracer and 33 min for the human tracer. The clearance was monitored for 10 h during which the human tracer continued to be cleared more rapidly than the chicken tracer. The proportion of radioactivity appearing as low molecular weight degradation products increased with time. Acid gel permeation and reverse-phase HPLC of the binding protein-associated radioactivity demonstrated that the labelled IGF-I bound was intact IGF-I. Sephadex G-200 gel permeation chromatography of chicken plasma samples at pH 7·4 showed that the binding protein complex labelled in vivo with chicken IGF-I tracer had a molecular mass of 55 kDa. Furthermore, the tracer associated with the binding protein coeluted with the major peak of endogenous IGF-I, suggesting that the tracer was bound to the physiologically relevant binding protein. Similar results were obtained with Superose 12 chromatography. Human plasma chromatographed as expected with much of the immunoreactivity and the labelled IGF-I eluting with a mass of 150 kDa on both Sephadex G-200 and Superose 12 columns. Ligand blotting of plasma binding proteins with 125I-labelled IGF-I after first subjecting the samples to polyacrylamide gel electrophoresis and transfer to nitrocellulose confirmed that the predominant binding protein in chicken plasma differed from the major forms in human plasma.

Journal of Endocrinology (1990) 124, 361–370
Effects of passive immunization of growing guinea-pigs with an insulin-like growth factor-I monoclonal antibody
Authors: Kerr D. E. , Laarveld B. , Manns J. G.
Published: December 2008

The physiological importance of circulating as opposed to locally produced insulin-like growth factor-I (IGF-I) has not been determined. By using a passive immunoneutralization technique, our objectives were to evaluate the role of circulating IGF-I in the regulation of animal growth and pituitary GH content.

A monoclonal antibody (MAb) to IGF-I, generated in our laboratory, has an affinity (Ka) of 0·13 litres/pmol for recombinant human IGF-I (rhIGF-I). Cross-reactivities of recombinant des-tripeptide IGF-I and recombinant bovine IGF-II were approximately 40 and 8% respectively. This MAb inhibited binding of purified hIGF-I to human placental membranes. In a radioimmunoassay based on displacement of 125I-labelled rhIGF-I from the MAb, displacement curves generated with dilutions of acid–gel chromatography extracts of guinea-pig serum and rhIGF-I standards were parallel.

Twenty-four, 3-week-old male guinea-pigs were treated with the IGF-I MAb, a bovine herpes virus-I (BHV-I) MAb (control MAb) or vehicle (phosphate-buffered saline) (n = 8 per group). Treatments were administered i.p. every 3 days for 24 days at a dose of 20 mg/kg body weight. Blood was obtained on day 23 (48 h after treatment) and on day 25 (24 h after treatment). In a liquid-phase assay, serum from the IGF-I MAb-treated group bound 38 ± 8% (mean ± s.e.m.) (day 23) and 56 ± 7% (day 25) of an 125I-labelled rhIGF-I trace at a final dilution of 1:10 000. Because of the development of an anti-mouse immune response in the guinea-pigs, these parameters would probably have been much greater during the first 2 weeks of the trial. Of the total IGF-I in serum, 50 ± 5% and 61±4% could be immunoprecipitated with an excess of rabbit anti-mouse immunoglobulin in samples from days 23 and 25 respectively. Comparisons between the groups treated with IGF-I MAb and BHV-I MAb revealed no significant differences in whole animal growth rate, growth of individual tissues, or pituitary GH content. Mean serum concentrations of IGF-I were 69 and 99% greater in IGF-I MAb-treated group than in the BHV-I MAb-treated group on days 23 and 25 respectively. These differences probably resulted from an extension of the half-life of IGF-I in serum of animals treated with the IGF-I MAb.

The lack of effect of treatment with the IGF-I MAb suggests that local production of IGF-I is generally sufficient to maintain normal growth or that local production or activity of IGF-I is increased in a compensatory fashion.

Journal of Endocrinology (1990) 124, 403–415
Effect of the progesterone antagonist mifepristone on the hypothalamo-hypophysial-ovarian axis in rats
Authors: van der Schoot P. , Uilenbroek J. Th. J. , Slappendel E. J.
Published: December 2008

Treatment of female rats for 3 weeks with the antigestagen 1 1β-(4-dimethylaminophenyl)-17β-hydroxy-17α-(prop-1 -ynyl)-estra-4,9-dien-3-one (mifepristone) results in pituitary and ovarian enlargement. The present study dealt with the possible mechanism(s) of these responses.

Ovarian enlargement appeared to be dependent upon prolactin. In the absence of prolactin, during combined treatment with mifepristone and the dopamine agonist 2-Br-α-ergokryptine, ovarian growth was significantly suppressed. It was unclear why persistent hyperprolactinaemia, due to treatment with mifepristone, resulted in persistence of functionally active corpora lutea despite intermittent ovulation, while persistent hyperprolactinaemia due to ectopic pituitary grafts did not.

Pituitary enlargement appeared to be dependent upon the persistence of ovarian oestrogen secretion during the treatment period. Ovariectomy or lactation fully inhibited this response. Pituitary enlargement and prolactin secretion in ovariectomized rats in response to exogenous oestrogen (injections of oestradiol benzoate) were significantly enhanced by additional treatment with mifepristone. It is concluded that mifepristone facilitates the effect of oestrogen on pituitary lactotrophs, thereby enhancing pituitary growth.

Ovarian enlargement during treatment with mifepristone may be specific for rats due to the luteotrophic action of prolactin in these animals. Pituitary enlargement due to facilitation of oestrogen-induced pituitary growth may become a focus of attention when this or similar antigestagenic drugs are being used for prolonged periods in clinical trials, e.g. for limiting steroid-sensitive tumour growth.

Journal of Endocrinology (1990) 124, 425–432
Characterization of α1-adrenergic receptor subtypes linked to iodide efflux in rat FRTL cells
Authors: Shimura H. , Endo T. , Tsujimoto G. , Watanabe K. , Hashimoto K. , Onaya T.
Published: December 2008

We have characterized α1-adrenergic receptor subtypes in functional rat thyroid cells, FRTL, with relation to iodide efflux, and have also examined the effect of TSH on α1 receptor subtypes. FRTL cells grown in a medium containing 5 mU TSH/ml (6H cells) had five times the number of α1 receptors of those maintained in TSH-free medium (5H cells) (11·2 fmol/106 cells compared with 2·0 fmol/106 cells). Pretreatment with chlorethylclonidine (CEC; 10 μmol/l), which inactivates only α1b receptors, caused 98·8% and 97·0% decreases in the density of specific [3H]prazosin-binding sites in 5H and 6H cells respectively. LIGAND computer program analysis of the displacement curves for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane (WB4101) showed that FRTL cells contained mostly low-affinity WB4101 sites. Using the phenoxybenzamine inactivation method, we found a linear relationship between α1 receptor density and the cytosolic free Ca2+ concentration response in FRTL cells. Pre-exposure of intact FRTL cells to CEC caused a 98·7% decrease in noradrenaline-stimulated maximal increase in cytosolic free Ca2+. Also, CEC and 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8), but not nicardipine, inhibited noradrenaline-stimulated iodine efflux. The results suggest that FRTL cells contain mostly the α1b-adrenergic receptor subtype; that the α1b receptors mediate cytosolic free Ca2+ and iodide efflux responses, and that TSH enhances these responses by increasing the α1b receptor density without affecting the post-receptor mechanism.

Journal of Endocrinology (1990) 124, 433–441
Production of anti-idiotypic antisera to rat GH antibodies capable of binding to GH receptors and increasing body weight gain in hypophysectomized rats
Authors: Gardner M. J. , Morrison C. A. , Stevenson L. Q. , Flint D. J.
Published: December 2008

Anti-idiotypic antibodies to rat GH antibodies were produced in both sheep and mice and shown to be capable of mimicking GH by inhibiting 125I-labelled ovine GH (oGH) binding to sheep liver membranes. The sheep anti-idiotypes were characterized further and shown to (1) inhibit 125I-labelled oGH binding to oGH antibodies, (2) inhibit 125I-labelled oGH binding to rat adipocytes and (3) be incapable of inhibiting the binding of either 125I-labelled ovine prolactin or 125I-labelled bovine insulin to sheep liver membranes. This indicated that the antibodies were not limited to certain species or tissues, but were hormone specific. Finally, these anti-idiotypic antibodies were also capable of stimulating an increase in body weight gain in hypophysectomized rats, suggesting that they may be functional as well as structural mimics of GH, although the increased body weight gain was not accompanied by any increase in circulating concentrations of insulin-like growth factor-I.

Journal of Endocrinology (1990) 125, 53–59
Regulation by GH of insulin-like growth factor-I mRNA expression in rat epiphyseal growth plate as studied with in-situ hybridization
Authors: Nilsson A. , Carlsson B. , Isgaard J. , Isaksson O. G. P. , Rymo L.
Published: December 2008

Expression of insulin-like growth factor I (IGF-I) mRNA and its dependence on GH was studied in rat epiphyseal growth plate by in-situ hybridization. Methodological aspects of the technique were first evaluated on fixed sagittal cryosections from 28-day-old rat epiphyseal growth plates to ascertain specific hybridization. In-situ hybridization was compared on sections from 10- and 28-day-old rats and the GH-dependence was studied in 35-day-old hypophysectomized rats. Expression of IGF-I mRNA was apparent in chondrocytes of the proliferative, hypertrophic and early degenerative zones of the growth plate of 28-day-old rats. The epiphyseal growth plate from 10-day-old rats showed a weak hybridization signal compared with 28-day-old rats. In contrast, the mesenchymal cells of the periosteum of 10-day-old rats showed a rather strong hybridization signal. Hypophysectomy resulted in a reduction in hybridization signal and cell number of the growth plate compared with 35-day-old age-matched normal rats. GH-Replacement therapy (200 μg human GH s.c. every 4 h for 24 h) resulted in partially restored expression of IGF-I mRNA.

The present study has shown that the IGF-I gene is expressed in the rat epiphyseal growth plate chondrocytes and that the expression is dependent on GH. The results support a paracrine/autocrine role of IGF-I for the expression of the stimulatory effect of GH on longitudinal bone growth.

Journal of Endocrinology (1990) 125, 67–74
Possible site of action of 5-hydroperoxyeicosatetraenoic acid derived from arachidonic acid in ACTH-stimulated steroidogenesis in rat adrenal glands
Authors: Mikami K. , Omura M. , Tamura Y. , Yoshida S.
Published: December 2008

The site of action of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) in ACTH-induced stimulation of steroidogenesis was examined in rat adrenocortical fasciculata cells. Prior addition of AA861, a specific inhibitor of 5-lipoxygenase, had no significant effect on cyclic AMP-dependent protein kinase activity and cholesterol esterase activities, when stimulated by ACTH in adrenocortical cells, compared with that stimulated by ACTH alone. Cholesterol accumulation in the mitochondria of cells treated with ACTH and cycloheximide was also not altered by pretreatment with AA861. We found, however, that pregnenolone formation, stimulated by ACTH, decreased in a dose-dependent manner when cells were pretreated with AA861. The inhibition of ACTH-stimulated pregnenolone formation by treatment with AA861 was restored only by prior addition of 5-HPETE. Furthermore, addition of AA861 also did not affect the conversion of pregnenolone into corticosterone.

In conclusion, 5-HPETE may act at the level of the mitochondria in ACTH-induced steroidogenesis in rat adrenal fasciculata cells.

Journal of Endocrinology (1990) 125, 89–96
Differential responses of hypothalamic LHRH-I and -II to castration and gonadal steroid or tamoxifen treatment in cockerels
Authors: Wilson S. C. , Gladwell R. T. , Cunningham F. J.
Published: December 2008

Changes in the hypothalamic contents of LHRH-I and LHRH-II were determined in intact and castrated cockerels injected i.m. with gonadal steroids or tamoxifen. An increase in the plasma concentration of LH after castration was accompanied by a significant increase in the content of LHRH-I in the posterior hypothalamus (including the mediobasal hypothalamus and median eminence) which was reversed by oestradiol benzoate given on days 14 and 15 after castration. Under similar circumstances, testosterone propionate did not modify the hypothalamic content of LHRH-I, even though both steroids reduced the plasma concentrations of LH to levels below those of intact cockerels. Treatment of intact cockerels with oestradiol benzoate significantly increased the content of LHRH-I in the posterior hypothalamus, whilst testosterone propionate was again without effect. Tamoxifen significantly raised the plasma concentration of LH in intact cockerels and partially antagonized the suppressive effect of oestradiol benzoate and testosterone on LH secretion in castrated cockerels. However, an anti-oestrogenic effect of tamoxifen on the hypothalamic content of LHRH-I was not demonstrated. There was no evidence of any changes in the hypothalamic content of LHRH-II after castration, with or without gonadal steroid replacement. A change in the hypothalamic content of LHRH-I in response to manipulation of the steroid environment would imply an involvement of this peptide in the mechanism by which gonadal steroids regulate the release of LH. The absence of changes in the hypothalamic content of LHRH-II in the same circumstances suggest that it is not directly involved in the control of LH secretion by the gonadal steroid negative feedback loop.

Journal of Endocrinology (1990) 125, 139–146
Steroid, corticotrophin-releasing hormone, ACTH and prostaglandin interactions in the amnion and placenta of early pregnancy in man
Authors: Jones S. A. , Challis J. R. G.
Published: December 2008

Corticotrophin-releasing hormone (CRH) is produced by both the placenta and fetal membranes at term in man, and CRH mRNA has been detected in human placental tissue. The synthesis of CRH and its control during early pregnancy, however, have not been established, and the role of CRH produced in the placenta and fetal membranes is not known.

We examined whether amnion and placental tissue obtained between 12 and 15 weeks of gestation produced CRH in vitro, whether steroid modulation of output occurred and whether CRH affected prostaglandin (PG) output by the placenta and amnion.

Immunoreactive (ir) CRH output by amnion (2·8 ± 0·31 (s.e.m.) nmol/105 cells) was significantly (P<0·01) greater than that from placenta (1·76 ± 0·21 nmol/105 cells). Output of ir-CRH decreased in the presence of progesterone, but increased in the presence of cortisol and dexamethasone. There was no significant effect of progesterone or ir-CRH output by placental cells; however, ir-CRH output was increased in the presence of dexamethasone and cortisol. There was no significant effect of corticosterone on ir-CRH output by either amnion or placental cells. Both ACTH and CRH stimulated the output of PGE2 and PGF2α by amnion cells. In contrast, there was no significant effect of PGE2 output by placental cells maintained in the presence of either human CRH or ACTH. Output of PGF2α by placental cells was increased in the presence of both CRH and ACTH.

We conclude that both amnion and placental tissue produce CRH in early gestation, and that this output is modulated by steroids. CRH and ACTH stimulate PG output by amnion and placenta in early pregnancy, raising the possibility of local (autocrine and paracrine) interrelations in vivo.

Journal of Endocrinology (1990) 125, 153–159
Influence of chronic oestrogen treatment on the density of muscarinic cholinergic receptors and calcium channels in the rabbit uterus
Authors: Batra S.
Published: December 2008

Ovariectomized rabbits were treated for 1 or 8 weeks with oestrogen and the effects on uterine weight, muscarinic receptor density and nitrendipine-binding sites on myometrial membranes were studied. The wet weight of the uterus after 1 week of oestrogen treatment increased more than tenfold and increased further by approximately 50% after 8 weeks of oestrogen treatment. The muscarinic receptor density increased significantly after 1 week of oestrogen treatment. Although there was no further increase in receptor density with continuing oestrogen treatment up to 8 weeks, the muscarinic receptor affinity increased significantly. The density of nitrendipine-binding sites increased sixfold after 1 week of oestrogen treatment and did not change thereafter. Oestrogen treatment had no influence on the affinity of nitrendipine-binding sites. The increase by oestrogen in muscarinic receptor density and in nitrendipine-binding sites as shown by the present data is consistent with the generally reported increase in agonist-induced contractile response of the myometrium following oestrogen treatment.

Journal of Endocrinology (1990) 125, 185–189
The cytochrome P45017α (17α-hydroxylase/C17,20-lyase) activity of the junctional zone of the rat placenta
Authors: Johnson D. C. , Sen M.
Published: December 2008

The cytochrome P450 responsible for androgen synthesis by the placenta during the second half of pregnancy in the rat was studied in intact and hypophysectomized animals. The two activities of P45017α, 17α-hydroxylase and C17,20-lyase, were limited to the junctional zone.

C17,20-Lyase activity was greater with progesterone than with 17-hydroxyprogesterone as substrate. Although the apparent Michaelis constants were similar, progesterone had a higher maximum velocity than 17-hydroxyprogesterone. Regardless of substrate, C17,20-lyase activity was greater with NADPH than with NADH as an electron donor, and there was no additive effect using both cofactors.

Administration of human chorionic gonadotrophin (hCG; 10IU at 09.00 and 21.00 h on days 13 and 14 and at 09.00 h on day 15) to intact females resulted in more than a 50% reduction of enzyme activity when measured on day 15. The same dose of hCG given to hypophysectomized animals with delayed implantation, i.e. pituitary removal on day 3 and implantation induced by oestrone 5 days later, had no effect on placental enzyme activity, but increased that in the ovary. Administration of ovine LH by osmotic minipump (days 11–15) to intact females resulted in abortion in all animals. The same treatment to animals hypophysectomized on day 11 produced abortion in three of four rats; enzyme activity was greatly reduced in the single animal with placentas. In contrast, infusion of LH into hypophysectomized animals with delayed implantation increased placental enzyme activities. However, administration of 20IU pregnant mare's serum gonadotrophin to the hypophysectomized animals on day 11 produced a large increase in ovarian follicular development and ovarian P45017α activities, and resulted in abortion in three of five animals within 96 h.

The results indicate that the P45017α of the junctional zone of the rat placenta has characteristics similar, if not identical, to those of the ovary with regard to preference of substrate and electron donor. However the enzyme activity in the placenta did not increase to the same extent as that in the ovary when exposed to gonadotrophins with LH activity, suggesting that the control mechanisms may be different.

Journal of Endocrinology (1990) 125, 217–224
Available FSH receptors in adult rat testis in vivo
Authors: Yoon D. J. , Reggiardo D. , David R.
Published: December 2008

It has been suggested that the tight junctions formed between Sertoli cells during the peripubertal period constitute a barrier to circulating FSH in the adult testis, limiting its access to the lumen of the seminiferous tubules. There are also reports of FSH receptor-binding inhibitors. These obervations prompted us to study the extent of FSH receptor availability in vivo in the adult rat. Experimental rats were given an intracardiac injection of rat FSH (rFSH), and the occupied receptor was measured by radioimmunoassay of acid-released rFSH from the testis. In the saline-injected control animals, there were 247 fmol occupied FSH receptors/g testis, and as much as 1788 fmol of the unoccupied high-affinity receptors/g testis, as measured by in-vitro binding studies. After intracardiac injection of increasing amounts of rFSH (up to 606 pmol), receptor occupancy increased to a maximum plateau of only 448 fmol/g testis. In contrast, when rFSH was given by intratesticular injection in order to achieve pharmacological doses in the testis, the maximum binding was 662 fmol/g testis. Scatchard analysis of the in-vivo data revealed, however, that the maximum concentration of the high-affinity receptor was 452 fmol/g testis, a value concordant with the highest in-vivo binding observed in animals given intracardiac rFSH (448 fmol/g). A single injection of the hormone did not induce down-regulation of FSH receptors, regardless of the dose, whereas multiple injections of menotrophin were effective, at least to some extent. Despite the receptor loss, the immediate receptor availability was maintained, suggesting the presence of a receptor pool.

In conclusion, in-vivo binding of FSH to its high-affinity receptor is limited in adult rat testis, and the available receptor concentration appears to be regulated so as to maintain a constant level.

Journal of Endocrinology (1990) 125, 293–299
Enzyme-linked immunosorbent assay for human insulin-like growth factor-I using monoclonal and polyclonal antibodies with defined epitope recognition
Authors: Tamura K. , Kobayashi M. , Suzuki S. , Ishii Y. , Koyama S. , Yamada H. , Hashimoto K. , Niwa M. , Shibayama F.
Published: December 2008

Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1–25 μg/l, compared with the range of 1–50 μg/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.

Journal of Endocrinology (1990) 125, 327–335
Luteal steroidogenesis and regression in the rat: Effects of human chorionic gonadotrophin and phospholipase A2 on cells and plasma membranes
Authors: Greenhalgh E. A.
Published: December 2008

Progesterone production in vitro was measured in response to human chorionic gonadotrophin and phospholipase A2 using dispersed luteal cells from control and prostaglandin F2α (PGF2α)-treated rats. Highly purified luteal plasma membrane suspensions were prepared by partition distribution in dextranpolyethylene glycol, and membrane fluidity was measured by fluorescence polarization using the probe, trans-parinaric acid. Fluorescence polarization was highly dependent upon calcium, and was markedly affected by PGF2α treatment. No support was found for the theory that bulk membrane fluidity has a role in receptor or enzyme hindrance in the induction of luteal cell regression. However, membrane phase transitions induced by calcium interaction with anionic phospholipids and free radical damage may be involved in regression of the rat corpus luteum.

Journal of Endocrinology (1990) 125, 387–396
Luteal steroidogenesis and regression in the rat: Progesterone secretion and lipid peroxidation induced in luteal cells by human chorionic gonadotrophin, phospholipase A2 and prostaglandin F2α
Authors: Greenhalgh E. A.
Published: December 2008

Mid-luteal phase (control) and mid-luteal phase luteal cell suspensions from rats given prostaglandin F2α (PGF2α) to induce luteal regression and killed 24 h later (P24-regressed) were incubated at 37 °C in serum-free medium. Progesterone secretion and lipid peroxidation induced by treatment with human chorionic gonadotrophin (hCG) alone or hCG plus phospholipase A2 and PGF2α were measured. Differences were demonstrated in progesterone secretion and lipid peroxide measurements between control and P24-regressed cell suspensions. The experiments suggested that malondialdehyde formation may be a useful indicator of luteal regression and cell death.

Journal of Endocrinology (1990) 125, 397–402
Feedback effects of growth hormone on anterior pituitary function in chickens
Authors: Lea R. W. , Harvey S.
Published: December 2008

Circulating GH concentrations were suppressed 24 h after intracerebroventricular (i.c.v.) injection of native or recombinant DNA-derived chicken GH (rcGH) (10 μg in 4 μl vehicle) into the right lateral ventricles of conscious 6-week-old cockerels. Plasma concentrations of LH were suppressed within 1 h of i.c.v. administration of GH at doses of 1 or 10 μg, and remained suppressed for 24 h in birds injected with the highest concentration of GH. In contrast, concentrations of plasma prolactin were increased 24 h after i.c.v. administration of GH (10 μg). The i.v. administration of rcGH (at concentrations of 10 or 100 μg/kg) did not mimic the effects of i.c.v. injection of GH.

These results demonstrate central effects of GH on pituitary function in the domestic fowl.

Journal of Endocrinology (1990) 125, 409–415
Oxytocin has a role in gonadotrophin regulation in rats
Authors: Robinson G. , Evans J. J.
Published: December 2008

We previously demonstrated that oxytocin stimulates LH release from rat pituitary cells in vitro and advances follicular development and ovulation in mice in vivo. This study reports an investigation of rat LH levels following in-vivo administration of oxytocin. Injection of oxytocin (10 mIU/g, i.p.) to rats at 07.00, 08.00 and 09.00 h of pro-oestrus or at 09.00, 10.00 and 11.00 h of pro-oestrus advanced the onset of the LH surge (P<0.005) and attainment of peak concentrations of LH (P<0.02) in peripheral blood. On the other hand, the descending phase of the LH surge and the surge amplitude were not altered by oxytocin. Treatment at 05.00, 06.00 and 07.00 h of pro-oestrus or at 11.00, 12.00 and 13.00 h of pro-oestrus had no effect on the LH profile. A higher oxytocin dose (20 mIU/g) inhibited LH release when treatment was begun at 05.00, 07.00 or 09.00 h of pro-oestrus. A lower dose (5 mIU/g) was ineffective in altering LH concentrations. In addition, injections of oxytocin (10 mIU/g) at oestrus, metoestrus or dioestrus had no effect on the release of LH. Thus the efficacy of oxytocin in altering concentrations of LH was dose dependent and also critically affected by the day of the oestrous cycle and the time of pro-oestrus. Removal of endogenous oxytocin activity by the use of an oxytocin receptor antagonist abolished the pro-oestrous LH surge, indicating that oxytocin is a vital physiological component of the LH-releasing mechanism in rats. The study provides unequivocal evidence that oxytocin induces LH release in vivo, but the manifestation of oxytocin activity is dependent upon conditions of exposure.

Journal of Endocrinology (1990) 125, 425–432
The role of angiotensin-converting enzyme in the rat epididymis
Authors: Wong P. Y. D. , Uchendu C. N.
Published: December 2008

In order to investigate the role of renin angiotensin in the epididymis, angiotensin-converting enzyme (ACE) activity and angiotensin I (AI) and angiotensin II (AII) concentrations were measured in the male reproductive tract and blood serum of the rat. High ACE activity was detected in the rat epididymis, with a major part of the activity being associated with epididymal spermatozoa. When spermatozoa were prevented from entering the epididymis by efferent duct ligation, the ACE activity in the epididymis was greatly reduced. The epithelial cells lining the epididymal duct were also shown to possess ACE activity which was dependent upon circulating androgens.

Treatment of male rats with captopril at a single oral dose (20 mg/kg) significantly inhibited the ACE activity in the blood serum but had no effect on the activity of the epididymal fluid. The intraluminal ACE was protected from the circulating captopril by the blood–epididymis barrier. Long-term treatment with captopril (20 mg/kg per day, 8 weeks), however, caused an increase in blood serum ACE activity but was without effect on intraluminal ACE. The fertility and fecundity of male rats after treatment were apparently normal.

The concentrations of AI and AII were high in the epididymal plasma and epididymal cell when compared with the respective concentrations in blood serum. The intraluminal AII concentration found (13 nmol/l) was close to the threshold concentrations that stimulate anion (and fluid) secretion in cultured epididymal epithelium in vitro. The high intraluminal AII concentration could not have been derived from the testicular fluid or spermatozoa since the rete testis fluid and sperm contained little AII. When spermatozoa were prevented from entering the epididymis by efferent duct ligation, the AII concentration of the epididymal plasma was almost completely abolished, indicating that intraluminal AII was formed endogenously in the epididymal lumen by sperm ACE. We propose that ACE released by epididymal spermatozoa converts AI (formed from the epididymal epithelial cells) to AII which plays a paracrine role in regulating electrolyte and fluid transport through apical membrane angiotensin receptors.

Journal of Endocrinology (1990) 125, 457–465
Tri-iodothyronine inhibition of thyrotrophin-releasing hormone-induced growth hormone release from the chicken adenohypophysis in vitro
Authors: Harvey S.
Published: December 2008

Tri-iodothyronine (T3) had no effect on the basal level of GH release from chicken hemipituitary glands perifused in vitro. The GH response to TRH was, however, markedly suppressed following exposure to T3. Suppression of TRH-stimulated GH secretion was observed after a 2-h preincubation with T3, and was induced, in a dose-related way, by 0·01–10 μmol T3/l. Exposure to T3 also reduced the effectiveness of TRH, at concentrations of 0·001–10 μg/ml, to stimulate GH release.

These results demonstrate that, in addition to a hypothalamic site of action, T3 is likely to suppress GH secretion in vivo by direct effects on pituitary GH release.

Journal of Endocrinology (1990) 126, 75–81
Effects of prostaglandin F2α and other potential secretagogues on oxytocin secretion and second messenger metabolism in the ovine corpus luteum in vitro
Authors: McCann T. J. , Flint A. P. F.
Published: December 2008

Release of oxytocin by sliced or minced sheep luteal tissue in vitro was stimulated up to 1·6- and 2·3-fold by arachidonic acid and the calcium ionophore A23187 respectively. Prostaglandin (PG) F2α and the PGF2α analogue cloprostenol, and other potential agonists known to be active in vivo, including noradrenaline and acetylcholine, were ineffective, as was the phorbol ester tetradecanoylphorbol acetate (TPA). The ineffectiveness of PGF2α was not due to a general unresponsiveness of the tissue in vitro, as PGF2α reduced LH stimulation of tissue concentrations of cyclic AMP and activated inositol lipid hydrolysis. The effect of arachidonic acid was accompanied by release from the tissue of the cytosolic enzyme lactate dehydrogenase (at arachidonic acid concentrations below those required to release oxytocin) and its effect on oxytocin and lactate dehydrogenase release was mimicked by oleic and linolenic acids; arachidonic acid was concluded to act by a non-physiological physicochemical effect without conversion to an eicosanoid. As PGF2α in vitro is known to raise intracellular Ca2+ concentrations in the large luteal cells that secrete oxytocin, and as A23187 stimulates oxytocin release in vitro in the presence and absence of TPA, it is concluded that in-vitro incubation results in an artifactual blockade of the oxytocin-releasing action of PGF2α at an unidentified point distal to the effect on intracellular Ca2+.

Journal of Endocrinology (1990) 126, 89–98