bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of phorbol ester on protein kinase C activity and effects of depletion of its activity on thyrotrophin, forskolin and 8′-bromoadenosine 3′,5′-cyclic monophosphate-induced [3H]thymidine incorporation in rat FRTL-5 cells
Authors: Akiguchi I. , Izumi M. , Nagataki S.
Published: December 2008

Protein kinase C (PKC) activity was measured in rat FRTL-5 cells cultured in medium containing insulin and transferrin (2H medium), in cells treated with phorbol 12,13-dibutyrate (PDBu), an activator of PKC, and in cells treated with 4α-12,13-phorbol didecanoate (4α-PDD), an inactive phorbol ester, in order to study the translocation and down-regulation of its activity. Provided the PDBu induced translocation and down-regulation of PKC activity, we used the down-regulated cells to study the roles played by PKC in DNA synthesis induced by stimuli which increase cyclic AMP (cAMP) and a cAMP analogue. Cytosolic preparations obtained from cells cultured in 2H media were fractionated on a DEAE-cellulose column, and fractions were assayed for PKC activity. PKC activity was eluted at concentrations between 0·06 and 0·15 mol NaCl/l with a peak at 0·1 mol/l. Exposure of cells to PDBu (100 ng/ml) resulted in 53% loss of initial PKC activity in the cytosolic fraction in concert with reciprocal augmentation of PKC activity in the particulate fraction that peaked at 321% of the initial level at 15 min of exposure.

When cells were stimulated with 100 ng PDBu/ml for 15 min, three- and 1·9-fold greater PKC activity appeared in the particular and total cellular (cytosol plus particulate) fractions respectively than was the case for stimulation with 10 ng PDBu/ml. The total cellular PKC activity transiently increased at 15 min to 137% and subsequently decreased to 74, 40 and 25% of the initial level at 1, 12 and 24 h respectively. However, translocation of PKC activity to the particulate fraction was not observed in cells treated for 15 min with either 10 or 100 ng 4α-PDD/ml and total cellular PKC activity was modestly reduced, to 97% of the initial level in cells exposed to 4α-PDD (100 ng/ml) for 24 h.

[3H]Thymidine incorporation was measured in cells whose PKC activity was stimulated by TSH, forskolin or 8-bromoadenosine cAMP simultaneously with PDBu (100 ng/ml), and also in cells whose PKC activity had been down-regulated by pretreatment with PDBu prior to the addition of each stimulant. The [3H]thymidine incorporation induced by each of the stimulants was inhibited in cells whose PKC activity was subjected to simultaneous stimulation, but was enhanced in cells whose PKC activity had previously been depleted. This potentiating effect of PDBu pretreatment on subsequent TSH-induced [3H]thymidine incorporation was not observed in cells exposed to PDBu for less than 12 h, but was observed in cells exposed for 24 h or longer.

These findings support the hypothesis that PKC regulates TSH-induced DNA synthesis in an inhibitory fashion at sites both proximal and distal to the cAMP generation process in FRTL-5 cells, and that a critical amount of activity must be present for this inhibition to occur.

Journal of Endocrinology (1993) 138, 379–389
The influence of endocrine factors on the serum concentrations of insulin-like growth factor-I (IGF-I) and IGF-binding proteins
Authors: Sugisaki T. , Yamada T. , Takamatsu K. , Noguchi T.
Published: December 2008

Serum insulin-like growth factor-I (IGF-I) levels in lit (isolated GH deficiency), dw (panhypopituitarism), hyt (hypothyroidism) and cog (congenital goiter) mutant mice were found to be significantly lower than in control mice. In addition, liver and kidney IGF-I concentrations in mutants were also found to be significantly reduced compared with controls. These differences in IGF-I concentration were not observed in pg (genetic IGF receptor or post-receptor defect?) mice. These results indicated that serum IGF-I production is induced by thyroid hormones as well as by GH. Western blot analysis of serum IGF-binding protein (IGFBP) fractions revealed the following differences among the five mutants: the triplet of IGFBP-3 (42, 45 and 49 kDa) and IGFBP-4 (24 kDa) levels were significantly reduced. IGFBP-2 (32 kDa) levels were also reduced in the lit, hyt and cog mice, although the level in serum of dw mice was found to be greatly elevated. No differences in serum IGFBP levels were found in the pg mouse. Consequently, the ratio of IGFBP-3%: IGFBP-2% (the percentage of IGFBP-3 fraction of total IGFBP (IGFBP-3%) divided by that of IGFBP-2 (IGFBP-2%)) was decreased in GH-deficient mice but increased in hypothyroid mice, suggesting that IGFBP-3 and IGFBP-2 production in the liver is induced by GH and thyroid hormones respectively.

Journal of Endocrinology (1993) 138, 467–477
Tumour necrosis factor-α modulates oestradiol responsiveness of MCF-7 breast cancer cells in vitro
Authors: Danforth D. N. , Sgagias M. K.
Published: December 2008

We studied the effect of tumour necrosis factor-α (TNF) on oestradiol regulation of growth and metabolism of MCF-7 breast cancer cells to determine whether TNF altered the oestradiol responsivness of these cells. We found that TNF antagonized oestradiol stimulation of cell growth in a dose-dependent manner, with partial inhibition at 1·0 U/ml and complete inhibition at 1000 U/ml. TNF inhibited cell cycle progression, increasing cells in the G0G1 phase and blocking oestradiol-stimulated progression into the S phase. We examined the effect of TNF on three oestrogen-regulated proteins, the oestrogen receptor (ER), the progesterone receptor (PR) and insulin-like growth factor-I (IGF-I). TNF down-regulated the ER and up-regulated the PR. Both of these processes were enhanced by the addition of oestradiol. The effects of TNF on the ER and PR were dose-dependent and occurred without a change in the Kd of the receptor. TNF did not change the respective steady-state mRNA levels. In addition, TNF did not alter secretion of IGF-I either in the absence or presence of oestradiol, indicating that the effects of TNF on oestrogen-regulated proteins is selective. These findings indicate an important interaction between the immune and endocrine systems. The cytokine TNF has a prominent effect on oestradiol stimulation of MCF-7 cells, blocking its proliferative response and enhancing certain metabolic effects. These actions may be mediated in part through modulation of the ER, although other pathways appear to be involved.

Journal of Endocrinology (1993) 138, 517–528
Androgen regulation of circulating insulin-like growth factor-I during puberty in male hypogonadal mice
Authors: Crawford B. A. , Singh J. , Simpson J. M. , Handelsman D. J.
Published: December 2008

This study aimed at determining the relationship of sex steroids, particularly in the perinatal period, to the pubertal insulin-like growth factor-I (IGF-I) surge in male mice. We used hypogonadal (hpg) mice, which have a major deletion in the gonadotrophin-releasing hormone (GnRH) gene, in order to have a model lacking all GnRH-induced gonadotrophin and sex steroid secretion throughout pre- and postnatal life. Cross-sectional data on body weights and weights of testes, seminal vesicles, kidneys, liver and spleen from 9 to 77 days of age were obtained in male hpg, heterozygous (Hz) and homozygous normal (N/N) littermates (n = 75–78/group). These data did not reveal any difference between Hz and N/N mice. Hpg mice had decreased body weights which by 70–77 days of age were approximately 18% less than normal controls. Testes and seminal vesicles of hpg mice did not demonstrate any significant postnatal growth. Relative to body weight, kidney weights were also markedly reduced in hpg mice (P<0·0001), deviating significantly from normal by 28–35 days of age, reflecting the impact of androgen deficiency on a non-reproductive organ. From the cross-sectional data it was concluded that puberty commenced soon after weaning (21 days) in the male and that maturity was achieved within 4–5 weeks. Longitudinal study showed that, compared with normal controls, untreated hpg mice had an exaggerated pubertal IGF-I surge (P<0·005) which peaked in mid-puberty. This, together with their reduced body weights (P<0·05), were normalized by treatment from 21 to 70 days of age with two 1 cm s.c. implants of testosterone (n=6) or dihydrotestosterone (n=7). There was no difference in IGF-I levels or in weights of testes, seminal vesicles, kidney, liver or spleen between testosterone and dihydrotestosterone treatments (P>0·05). Prolonged high levels of androgen also restored testicular and seminal vesicle weights to 40% of phenotypically normal controls, while kidney, liver and spleen weights were also significantly increased. The pubertal IGF-I surge in mice does not, therefore, require androgens in either the pre- or postnatal periods, and it is exaggerated in androgen-deficient male mice and dampened to normal regardless of aromatization.

Journal of Endocrinology (1993) 139, 57–65
Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on the proliferation of bovine granulosa cells in vitro
Authors: Gong J. G. , McBride D. , Bramley T. A. , Webb R.
Published: December 2008

Treatment of heifers with recombinant bovine somatotrophin (BST) significantly increases the population of small ovarian follicles and peripheral concentrations of somatotrophin, insulin-like growth factor-I (IGF-I) and insulin. To investigate the possible mechanism(s) involved in the action of BST on ovarian follicles, the effects of BST, IGF-I and insulin, given alone or in combination with either FSH or LH, on the proliferation of bovine granulosa cells in vitro were examined using a serum-free culture system. Bovine granulosa cells were obtained from antral follicles classified into three size categories according to diameter: small <5 mm; medium-sized 5–10 mm and large >10 mm. The proliferation of granulosa cells was assessed by the incorporation of [3H]thymidine into the cultured cells.

Both FSH and LH (1–1000 ng/ml) inhibited the proliferation of bovine granulosa cells obtained from all three size classes of follicles in a dose-dependent manner. BST, at doses ranging from 1 to 1000 ng/ml, had no effect on the proliferation of granulosa cells from small and medium-sized follicles, but inhibited the division of granulosa cells from large follicles in a dose-dependent manner. Treatment with either IGF-I (10–3000 ng/ml) or insulin (0·5–1000 ng/ml) stimulated, in a dose-dependent manner, the proliferation of granulosa cells obtained from all three size categories of follicles. No synergistic interaction between BST (30 ng/ml) and either FSH (50 ng/ml) or LH (5 ng/ml) was observed in granulosa cells from all three size classes of follicles. In contrast, physiological concentrations of both IGF-I (100 ng/ml) and insulin (1 ng/ml) acted in synergy with both FSH (50 ng/ml) and LH (5 ng/ml) to stimulate the proliferation of granulosa cells from small follicles, whilst no such synergistic interactions were observed in granulosa cells from medium-sized and large follicles.

It was concluded that the increase in the number of small ovarian follicles induced by BST treatment in heifers may be mediated by increased peripheral concentrations of IGF-I and/or insulin, possibly acting in synergy with gonadotrophins. Furthermore, insulin probably acts through its own receptor rather than acting via the type-I IGF receptor, as it can stimulate the proliferation of bovine granulosa cells at physiological concentrations.

Journal of Endocrinology (1993) 139, 67–75
Interactions between growth hormone and nutrition in hypophysectomized rats: body composition and production of insulin-like growth factor-I
Authors: Bates P. C. , Loughna P. T. , Pell J. M. , Schulster D. , Millward D. J.
Published: December 2008

Hypophysectomy of adult rats results in a loss of body growth which can be reversed by treatment with GH. The increased growth caused by administration of GH is accompanied by an increase in food consumption. The effects of GH and interactions with nutrition were investigated by treating hypophysectomized rats with GH and either providing unrestricted food or preventing the increased food consumption by pair-feeding with the same intake as that of the hypophysectomized animals.

Over the 7-day experimental period, the GH-treated animals grew significantly when food was available ad libitum but did not gain body weight when an increase in food intake was prevented. However, there was a significant interaction between GH and nutrition on body composition; GH significantly decreased body fat and increased the protein: fat ratio only in the animals with the restricted intake. Gastrocnemius muscle weight was increased by GH regardless of food intake, but heart weight did not increase and liver weight was actually decreased by GH teatment when food intake was restricted. Serum concentrations of insulin and insulin-like growth factor-I (IGF-I) were increased by GH in the rats with food available ad libitum but not in the pair-fed rats. However, the liver concentration of IGF-I and its mRNA were increased by GH although the increase in IGF-I mRNA was modulated by the restricted food intake. The decreased weight of the liver in the pair-fed GH-treated rats, despite the increase in IGF-I mRNA, suggests that IGF-I does not influence liver growth. In the gastrocnemius muscle, however, GH increased IGF-I mRNA concentration similarly in both rats with food available ad libitum and in pair-fed rats. Decreased nutrition therefore modulated the action of GH but emphasized its nutrient partitioning effect, thus increasing the anabolic drive towards skeletal muscle growth; this appeared to be mediated by the local production of IGF-I within the muscle.

Journal of Endocrinology (1993) 139, 117–126
Rat placental lactogen-I binds to the choroid plexus and hypothalamus of the pregnant rat
Authors: Pihoker C. , Robertson M. C. , Freemark M.
Published: December 2008

Recent findings suggest that placental lactogen has a role in the regulation of hypothalamic function during pregnancy. To explore the mechanisms by which placental hormones may exert effects in the maternal central nervous system, we have examined the binding of rat placental lactogen-I (rPL-I) to brain slices from pregnant rats at mid- and late gestation. The binding of rPL-I to maternal rat brain was compared with that of human GH (hGH). Radiolabelled rPL-I bound specifically to ependymal cells of the choroid plexus in the lateral ventricles and in the roof of the third ventricle. The binding of 125I-labelled rPL-I was inhibited by unlabelled rPL-I, hGH or rat prolactin but not by rat GH, indicating that rPL-I and rat prolactin interact with a common binding site in maternal rat brain. Radiolabelled hGH bound to the choroid plexus and to ependymal cells lining the third ventricle in the region of the arcuate nucleus. In addition, hGH bound specifically to the ventromedial nuclei and to the medial preoptic area of the hypothalamus. The binding of radiolabelled hGH to all brain regions was inhibited by unlabelled rPL-I as well as hGH, indicating that rPL-I competes for lactogenic binding sites in the hypothalamus as well as the choroid plexus of the pregnant rat. These findings suggest potential mechanisms by which placental hormones may exert direct effects on the maternal central nervous system during pregnancy. The precise functions and roles of the PL-I binding sites in maternal choroid plexus and hypothalamus remain to be explored.

Journal of Endocrinology (1993) 139, 235–242
Nutritional regulation of insulin-like growth factor-I mRNA expression in salmon tissues
Authors: Duan C. , Plisetskaya E. M.
Published: December 2008

In salmonids, nutritional insufficiency leads to retarded growth and reduced hepatic GH receptors, but increased circulating GH levels. To understand the endocrine mechanism underlying the retarded growth in starved fish better, we investigated the effect of food deprivation and refeeding on circulating levels of GH and insulin, as well as insulin-like growth factor-I (IGF-I) mRNA expression in different tissues of juvenile coho salmon (Oncorhynchus kisutch). Deprivation of food for 2–4 weeks resulted in cessation of growth and a significant decrease in condition factor (an indicator of fish body shape). No difference in circulating insulin or glucose levels were found between starved and fed fish, whereas starvation increased the plasma GH levels. After 4 weeks of starvation, the plasma GH level rose to 9 ng/ml, which was four times as high as that of the fed fish. In spite of elevated circulating GH, hepatic IGF-I mRNA levels were significantly reduced after 4 weeks of starvation. No significant difference in IGF-I mRNA levels of fed and starved fish was found in other tissues, including kidney, spleen, ovary, gill filament and gut. Two weeks of refeeding significantly increased hepatic IGF-I mRNA levels and growth and reduced plasma GH levels. These results suggest that food deprivation primarily reduces IGF-I mRNA expression in the liver which results, most probably, in a decline in systemic IGF-I levels and consequently leads to the retarded growth of salmon.

Journal of Endocrinology (1993) 139, 243–252
Prolonged intermittent but not continuous administration of oestradiol-17β increases bone volume in the rat
Authors: Tobias J. H. , Gallagher A. , Chambers T. J.
Published: December 2008

We have previously found that administration of oestradiol-17β (OE2) to intact adult female rats of 19 days stimulates cancellous bone formation. However, this effect is not observed following longer periods of OE2 treatment, suggesting that the responsiveness of the skeleton to oestrogen's anabolic action is reduced after prolonged administration. A possible explanation for this is that oestrogen also suppresses bone resorption, which is an important stimulus for bone formation. We therefore investigated the effect of omitting OE2 for short periods, on the proximal tibial metaphysis of intact female rats. We found that, unlike continuous treatment with OE2 (40 pg/kg) for 56 days, omission of OE2 for 4 days out of every 20 was associated with a significant increase in cancellous bone volume. Although continuous and intermittent OE2 were both associated with a reduction in osteoclast surface, a decrease in the proportion of double fluorochrome-labelled surface was only seen after continuous OE2 treatment. We then studied the effects of longer periods of OE2 omission by giving OE2 (40 pg/kg) for three repeated cycles of: (1) OE2 for 16 days/vehicle for 4 days, (2) OE2 for 12 days/vehicle for 8 days, (3) OE2 for 8 days/vehicle for 12 days, or (4) OE2 for 4 days/vehicle for 16 days. We found a significant increase in cancellous bone volume when OE2 was stopped for either 4 or 8 days at a time. However, longer periods of OE2 omission did not affect bone volume, possibly because these were associated with an increase in bone resorption and/or a reduction in bone formation during the OE2-free period. In conclusion, we observed an increase in cancellous bone volume after prolonged treatment with oestrogen only if OE2 was omitted for short periods. This may be due, at least in part, to bone formation being maintained at a higher rate by such treatment than by either continuous OE2 administration or by intermittent administration where OE2 is discontinued for longer periods.

Journal of Endocrinology (1993) 139, 267–273
Stimulation of placental, fetal and neonatal growth by thyroxine administration to pregnant rats
Authors: Spencer G. S. G. , Robinson G. M.
Published: December 2008

The effects of daily thyroxine (T4) administration to pregnant rats on the growth of their fetuses and placentae were examined. Additional trials examined the effect of such treatment on the postnatal growth of the offspring. Injection of 10 μg T4 into the mother caused a tenfold elevation in maternal plasma T4 concentration, which remained elevated for more than 12 h, but returned to basal levels by 24 h after injection. Despite the increase in maternal T4 concentrations, no increase in fetal plasma T4 was found. A greater weight of both the fetuses (20·1%; P<0·001) and placentae (14·6%; P<0·05) was found in the young of T4-treated mothers compared with those from control mothers at delivery by hysterotomy on day 20 of gestation. In trials where mothers delivered naturally, birth weights of the young from mothers treated with T4 (10, 20 or 50 μg daily) were also significantly heavier than controls, and continued to grow faster postnatally. These data suggest that maternal thyroid hormone status is important for fetal growth, and further indicate that stimulation of the thyroid hormone axis during pregnancy could provide a method for improving postnatal growth.

Journal of Endocrinology (1993) 139, 275–279
Serum unmasks the binding of thyroid-stimulating hormone to endogenous and transfected receptors: evidence for a soluble form of the receptor in human thyroid
Authors: Willey K. P. , Hunt N. , Abend N. , Northemann W. , Ivell R. , Leidenberger F.
Published: December 2008

A specific homologous radioligand receptor assay for thyroid-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30 000–100 000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSHbinding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0·2 μm filter and, upon molecular sieving, had an apparent size of 300 000–1 000 000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.

Journal of Endocrinology (1993) 139, 317–328
Effects of growth hormone and an antiserum to rat growth hormone on serum IGF-I and muscle protein synthesis and accretion in the rat
Authors: Palmer R. M. , Flint D. J. , MacRae J. C. , Fairhurst F. E. , Bruce L. A. , Mackie S. C. , Lobley G. E.
Published: December 2008

Rats were injected twice daily for up to 10 days with GH or with a polyclonal antiserum to rat GH, commencing at 21–22 days of age.

Administration of bovine or human GH (1 mg/day) improved whole body growth rates by 22% and 29% respectively. Plantaris muscle mass was also increased, by 7 and 14% respectively. Anti-GH injected twice daily resulted in a 7% decrease in body weight at 4 days and a 10% reduction by 10 days. Similar decreases were observed in the total protein content of plantaris and soleus muscles. The decrease in the fractional rate of protein synthesis was proportionately greater than the decline in protein content in plantaris muscle whereas in the soleus no change in the rate of protein synthesis was observed, suggesting that the effect on this muscle was due to an increase in the rate of protein degradation.

Serum total IGF-I was unchanged by treatment with either GH or anti-GH while the amount of hepatic IGF-I mRNA was also unaffected by anti-GH injection. These data are consistent with a direct effect of GH or an effect mediated by an autocrine/paracrine mechanism of action on muscle but do not support a role for serum total IGF-I as an endocrine mediator of GH action.

Journal of Endocrinology (1993) 139, 395–401
Regulation of metabolic water and protein compartments by insulin-like growth factor-I and testosterone in growth hormone-deficient lit/lit mice
Authors: Donahue L. R. , Watson G. , Beamer W. G.
Published: December 2008

Insulin-like growth factor-I (IGF-I) and testosterone are major hormonal regulators of protein metabolism. We chose genetically GH-deficient little (lit/lit) mice to test whether these anabolic hormones act independently or in concert with each other to stimulate protein metabolism. Hormones were administered for 14 days at constant rates to 14-week-old lit/lit female mice, IGF-I was infused via mini-osmotic pumps at 30 μg/day and testosterone was administered using 30 mg pellets. Food consumption was measured during the experimental period, and at the end we measured: (a) serum IGF-I, IGF-I-binding proteins (IGFBPs) and blood urea nitrogen (BUN); (b) body and musculo-skeletal carcass weights; (c) musculo-skeletal carcass water, fat, protein and mineral; and (d) selected organ weights plus protein and DNA contents.

We found that both of these growth-stimulatory hormones, IGF-I and testosterone, alone and in combination, had anabolic effects on different metabolic compartments in specific target organs. The most unexpected finding in this study was that the IGF-I-induced increase in musculo-skeletal carcass weight arose solely from increased water, revealing the importance of this compartment as an early target of IGF-I action. Other effects caused specifically by IGF-I, but not testosterone, included increases in serum IGFBP-3, body weight and spleen weight. The specific effect of testosterone, but not IGF-I, was to increase serum IGFBP-2. Independent effects were induced by each hormone alone for kidney and spleen weight, kidney and spleen protein content and BUN. Synergistic effects of IGF-I plus testosterone were observed for the musculo-skeletal preparation, organ weights (liver, kidney, heart and adrenal), liver protein and kidney DNA content. Food consumption did not differ among experimental groups. We conclude that in nutritionally replete GH-deficient lit/lit female mice, IGF-I regulates both water and protein metabolism in an organ-specific manner, whereas the effect of testosterone in specific organs is limited to protein metabolism.

Journal of Endocrinology (1993) 139, 431–439
Effects of dietary recombinant human insulin-like growth factor-I on concentrations of hormones and growth factors in the blood of newborn calves
Authors: Baumrucker C R , Blum J W
Published: December 2008

Colostrum is rich in insulin-like growth factor-I (IGF-I) and IGF-II and the dietary effects of recombinant human IGF-I (rhIGF-I) on the newborn are of interest. The objective of this study was to examine the effects of dietary rhIGF-I upon selected hormones and growth factors in the blood. Calves were fed for the first 2 days of life with one of three experimental diets: (1) milk replacer plus isolated colostrum-derived globulin (MR−), (2) as (1) plus 98 μmol rhIGF-I/l (MR+) or (3) pooled cow colostrum. Thereafter, all animals received only milk replacer at 5% of body weight/feeding twice a day with only treatment 2 having the continued addition of 98 μmol rhIGF-I/l until completion of the experiment 7 days after birth. Radioimmunoassays for insulin, prolactin, IGF-I, IGF-II, GH, l-thyroxine, 3,5,3′-l-triiodothyroline and cortisol were conducted. With the exception of GH, all hormones and growth factors examined showed some form of dietary effect, but many were transient, changing only with the first feeding. Both insulin and prolactin concentrations exhibited a transient increase in blood at the first feeding, but insulin increased with the MR− treatment whereas prolactin increased with the MR+ treatment. Total IGF-I concentration in blood did not show any diet-induced changes for the first 4 days, but thereafter a rise in blood concentrations of IGF-I was observed. These data indirectly support the hypothesis that dietary IGF-I may be absorbed and causes transient systemic effects in the newborn calf.

Journal of Endocrinology (1994) 140, 15–21
Over-expression of oxytocin in the testes of a transgenic mouse model
Authors: Ang H-L , Ivell R , Walther N , Nicholson H , Ungefroren H , Millar M , Carter D , Murphy D
Published: December 2008

The bovine oxytocin gene has been expressed in the testes of two independent transgenic mouse lines. Hybridization and RNase protection analysis showed that the oxytocin transgene was transcribed from the normal functional promoter in the Sertoli cells of the seminiferous tubules in a developmentally regulated manner. Immunohistochemistry indicated that both oxytocin and neurophysin epitopes were expressed together in the Sertoli cells at stages I–V and X–XII of the cycle of the seminiferous epithelium. Furthermore, analysis with high- performance liquid chromatography showed that there was a tenfold increase in the amount of amidated oxytocin present in testicular extracts from the transgenic mice. However, there appeared to be no detectable effect of this overproduction of hormone on testicular morphology or fertility parameters. A significant decrease by 50% was detected only in the levels of intratesticular testosterone and dihydrotestosterone. The results point to a local paracrine role for oxytocin in the modulation of Leydig cell function.

Journal of Endocrinology (1994) 140, 53–62
Role of endogenous atrial natriuretic peptide in volume expansion diuresis and natriuresis of the Pekin duck
Authors: Gray D A
Published: December 2008

Polyclonal antibodies raised in a rabbit against avian atrial natriuretic peptide (ANP) were shown to reduce circulating endogenous ANP levels in Pekin ducks by more than 90%, and were subsequently used to investigate the role of this peptide in volume expansion diuresis and natriuresis. Conscious birds, undergoing a steady-state diuresis and natriuresis maintained by an i.v. infusion of hypotonic saline at a rate of 0·7 ml/min, responded to ANP antiserum (anti-ANP) with an immediate 30% reduction in urine flow rate and sodium excretion which lasted for about 30 min. Plasma arginine vasotocin levels were not changed by anti-ANP whereas circulating angiotensin II concentrations increased immediately following the administration of anti-ANP. Serum from non-immunized normal rabbits produced no changes in the renal and plasma parameters monitored.

The results show that the high circulating levels of endogenous ANP associated with volume expansion promote renal salt and fluid excretion and thus have a major physiological role in avian volume homeostasis.

Journal of Endocrinology (1994) 140, 85–90
Free α subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains
Authors: Dirnhofer S , Lechner O , Madersbacher S , Klieber R , de Leeuw R , Wick G , Berger P
Published: December 2008

Immunochemical studies were undertaken to identify surface-orientated epitopes of the free α subunit of human chorionic gonadotrophin (hCG-α) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-α, an enzymatically digested hCG-α subunit, and a reduced and alkylated hCG-α preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-α, seven different epitopes (α1–α7), arranged in four spatially distinct domains, could be distinguished: A, α1,2,4; B, α3,5; C, α6; D, α7. The peptides spanning hCG-α(13–18), hCG-α(17–22) and hCG-α(33–42) appeared to contribute to the formation of epitopes α2, α4 and α6 respectively. Since epitope α6 is present only on the free non-assembled subunit of different species, we concluded that the region hCG-α(33–42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (α7) are destroyed by reducing and alkylating hCG-α. In contrast, chymotryptic digestion of hCG-α, leading to release of the heptapeptide hCG-α(41–47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-α epitopes by radioreceptor assay revealed that the three hCG-α peptides corresponding to epitopes α2, α4 and α6 did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (α1–α5), shared by hCG and hCG-α, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains.

Journal of Endocrinology (1994) 140, 145–154
Participation of the superior ovarian nerve in the regulation of compensatory ovarian hypertrophy: the effects of its section performed on each day of the oestrous cycle
Authors: Chávez R , Domínguez R
Published: December 2008

The effects were analysed of section of the superior ovarian nerve on compensatory ovulation and ovarian hypertrophy, in adult rats with the left or right ovaries extirpated during the oestrous cycle and autopsied 6 or 20 days later. Rats with hemiovariectomy or hemiovariectomy plus denervation recovered their oestrous cyclicity between 2 and 3 days after surgery. Six days after hemiovariectomy 14 out of 17 rats ovulated on the expected day of oestrus. All the animals were hemiovariectomized on the day of pro-oestrus. The mean ± s.e.m. number of ova shed was similar to the group of animals with both ovaries (7·8 ± 1·2 vs 9·5 ± 0·2 Compensatory ovarian hypertrophy was observed in the right ovary when left hemiovariectomy was performed on day 2 of dioestrus or pro-oestrus; similar results were observed in the left ovary when the right one was extirpated at oestrus or pro-oestrus. Section of the right superior ovarian nerve in left-hemiovariectomized rats caused a reduction in ovulation rate and number of ova released. Compensatory ovarian hypertrophy was modified in the opposite way by unilateral section of the superior ovarian nerve to the in situ ovary depending on the day of the cycle when hemiovariectomy was performed. Twenty days after treatment, ovulation rate, compensatory ovulation and ovarian hypertrophy were similar in both left- or right-hemiovariectomized rats. Compensatory ovarian hypertrophy increased in all animals with section of the superior ovarian nerve, except when hemiovariectomy was carried out at oestrus or the left ovary was extirpated on day 1 of dioestrus. The results suggest that modulation of compensatory ovarian hypertrophy by neural information arriving at the ovary by way of the superior ovarian nerve varies during the oestrous cycle.

Journal of Endocrinology (1994) 140, 197–201
Stimulatory effect of interleukin-1β on the hypothalamic-pituitary-adrenal axis of the rat: influence of age, gender and circulating sex steroids
Authors: Rivier C
Published: December 2008

The bilateral communication between the immune and neuroendocrine systems plays an essential role in modulating the adequate response of the hypothalamic-pituitary-adrenal (HPA) axis to the stimulatory influence of interleukins (ILs). It is thus reasonable to assume that inappropriate responses of the HPA axis to ILs might play a role in modulating the onset of pathological conditions such as infections. As part of our programme aimed at investigating the ability of ILs to release pro-opiomelanocortin-like peptides and corticosterone in rats exposed to alcohol, we observed that this stimulatory action appeared to be influenced by the gender of the animals. We therefore examined the ability of IL-1β, injected peripherally, to stimulate the HPA axis as a function of stage of sexual maturation and the presence or absence of circulating sex steroids.

In immature (21 to 22-day-old) rats, both males and females responded to the i.p. administration of 0·5 or 2·0 μg IL-1β/kg with statistically comparable increases in plasma ACTH levels. In contrast, females released significantly (P<0·01) more corticosterone in response to the lower dose of cytokine. Forty-day-old intact animals showed no sexual dimorphism in ACTH secretion, but the females again secreted significantly (P<0·05–0·01) more corticosterone. Gonadectomy, performed 7–8 days prior to the assay, increased the absolute amount of corticosterone released over a 60-min period. A noticeable dimorphism of the ACTH response to IL-1β became apparent in 70-day-old intact rats, with females secreting more ACTH than males. These females also released significantly more corticosterone than males under both resting and stimulated circumstances. In this age group, gonadectomy abolished the sex difference in terms of ACTH release, but augmented the total amount of corticosteroids secreted by both sexes, as well as increasing the sexual dimorphism.

These results suggest the presence of gender differences in the response of the HPA axis to IL-1β. While the sexual dimorphism of ACTH secretion appears to be dependent on circulating sex steroids, the sexually dimorphic adrenal response was retained following gonadectomy.

Journal of Endocrinology (1994) 140, 365–372
Effects of endothelin-1 and endothelin-3 on rat hypothalamic corticotrophin-releasing hormone and pituitary ACTH release in vitro
Authors: Calogero A E , Raiti F , Nicolosi G , Burrello N , D'Agata R , Mantero F
Published: December 2008

Endothelins (ETs) are potent vasoconstrictor peptides that also participate in the regulation of endocrine function. Indeed, immunoreactive ET, ET mRNA and ET receptors have been found in the brain and the pituitary gland and ETs stimulate arginine vasopressin, LH, FSH, TSH and gonadotrophin-releasing hormone and inhibit prolactin release in vitro. The present study was undertaken to evaluate the effects of ET-1 and ET-3, two members of this family, on corticotrophin-releasing hormone (CRH) release by explanted male rat hypothalami in vitro and on ACTH release by primary pituitary cell culture. ET-3 decreased basal CRH release in a concentration-related manner. The lowest effective concentration tested was 3 nmol/l but a more pronounced inhibitory effect was obtained at a concentration of 10 nmol/l. On the other hand, ET-1 did not have any detectable effect on basal CRH release. Neither ET-1 nor ET-3 had any effect on the release of CRH stimulated by potassium chloride. ET-1 increased basal ACTH release, whereas ET-3 did not have any effect. Both ET-1 and ET-3 suppressed the release of ACTH stimulated by 1 nmol CRH/l.

These data suggest that both ET-1 and ET-3 are able to modulate the hypothalamic-pituitary-adrenal axis function in vitro. However, they act at different levels and seem to have opposite effects. Indeed, while ET-1 stimulated pituitary ACTH release, ET-3, the peptide produced mainly in the brain, inhibited hypothalamic CRH release in vitro.

Journal of Endocrinology (1994) 140, 419–424
Low-dose cortisol infusion increases plasma corticosteroid-binding globulin (CBG) and the amount of hepatic CBG mRNA in fetal sheep on day 100 of gestation
Authors: Berdusco E T M , Milne W K , Challis J R G
Published: December 2008

Synthetic glucocorticoids stimulate the production of corticosteroid-binding globulin (CBG) by the liver of the sheep fetus near term (day 145). We have examined whether physiological changes in plasma cortisol alter plasma CBG concentrations, patterns of glycosylation and the amount of hepatic CBG mRNA at earlier times during pregnancy (day 100), prior to the activation of fetal hypothalamic-pituitary-adrenal function. Cortisol was infused into chronically catheterized sheep fetuses in amounts that raised the plasma cortisol concentration by about 15 nmol/l. This treatment resulted in a significant increase in the plasma corticosteroid-binding capacity and in the amount of CBG mRNA in the fetal liver, but did not alter the proportion of CBG retained using Concanavalin A chromatography. We conclude that the CBG gene in the liver of fetal sheep responds to physiological changes in plasma concentration of cortisol and we speculate that the rise in plasma CBG concentration is important in diminishing the negative feedback effect of circulating cortisol on the fetal pituitary and hypothalamus.

Journal of Endocrinology (1994) 140, 425–430
Secretory characteristics and phenotypic plasticity of growth hormone- and prolactin-producing cell lines
Authors: Kineman R D , Frawley L S
Published: December 2008

Considerable information regarding the regulation of GH and prolactin (PRL) release has been generated using pituitary cell lines as model systems. Inasmuch as these cultures have been derived from single cells by clonal selection it has frequently been assumed that they are composed of homogeneous populations of hormone secretors. However, experience with GH3 cells clearly demonstrates that such is not the case, since this GH- and PRL-producing line is comprised of a mixture of cells that are bihormonal, secrete only GH, or secrete neither hormone. Interestingly, the relative amount of these phenotypic subpopulations is not fixed, but can be altered by treatment with established regulators of GH and PRL secretion. This potential for secretory heterogeneity and phenotypic plasticity prompted us to examine the cellular composition of other commonly used GH- and/or PRL-secreting cell lines under control and treatment conditions. First, GH4C1, GH1, GC, MMQ and P0 cells were maintained according to established media and culture protocols and the relative abundance of GH and PRL secretors was assessed by reverse haemolytic plaque assays. As shown previously for GH3 cells, two cell lines were found to be functionally heterogeneous. Specifically, GH4C1 and GH1 were comprised of mixed populations of GH (25·9±1·1% and 51·3±6·5% (s.e.m.) respectively) and PRL (44·8±3·7 and 66·1 ±4·1% respectively) secretors. However, MMQ and GC cell cultures were relatively homogeneous with respect to hormone secretion in that the MMQ cells released PRL (72·2 ± 4·9%) but not GH, while GC cells released GH (93·6 ±1·4%) but not PRL. Similarly, P0 cell cultures contained predominantly GH-secreting cells (96·4±1·2%), but a few cells were detected that released PRL (1·1 ±0·1%). We then tested whether cortisol or oestradiol-17β (OE2) could alter the secretory composition of cell lines identified as reasonably homogeneous (GC, MMQ and P0) by treating cultures with steroids at doses ranging from 0·001 to 1000 nmol/l for up to 144 h. The presence of steroids in GC and MMQ lines did not affect the percentage of GH and PRL secretors. However, treatment of P0 cells with 0·001 to 1000 nmol OE2/l for 24, 48 and 144 h resulted in a dramatic increase in PRL cells when compared with controls. These increases occurred without alterations in GH percentages and in the absence of significant cell proliferation. In fact, OE2 suppressed the proliferation of P0 cells to 20% of the control rate (P<0·05), suggesting that OE2 induced pre-existing GH-secreting cells to release both GH and PRL. Taken together, these results clearly indicate that clonal origin does not guarantee homogeneity of function, and alteration in the culture environment can have profound effects on the secretory cell composition of acidophilic cell lines. These considerations have important implications for the interpretation of results generated with GH- and/or PRL-secreting cell lines.

Journal of Endocrinology (1994) 140, 455–463
Eel ventricular natriuretic peptide: isolation of a low molecular size form and characterization of plasma form by homologous radioimmunoassay
Authors: Takei Y , Takahashi A , Watanabe T X , Nakajima K , Ando K
Published: December 2008

Ventricular natriuretic peptide (VNP) with 25 amino acid residues was isolated from the low molecular weight fraction of acid extracts of eel cardiac ventricles. No other short forms of VNP were recovered from the fraction. This peptide was named eel VNP(1–25) because it was a C-terminally truncated form of the previously isolated eel VNP(1–36) As observed before with eel VNP(1–36), eel VNP(1–25) had a much higher (146-fold) vasodepressor activity than human atrial natriuretic peptide (ANP) in eels, but was a third to a half as active in rats with respect to vasodepressor and natriuretic activities. Eel VNP(1–25) was generally less potent than eel VNP(1–36) for vasodepressor and natriuretic effects.

A specific radioimmunoassay (RIA) has been developed for the measurement of eel VNP. The antiserum, raised against eel VNP(1–36), was highly specific and did not exhibit significant cross-reactivity with eel ANP and C-type natriuretic peptide, even though their amino acid sequences have more than 60% homology with that of eel VNP. The sensitivity of assay was 0·5 fmol/tube for eel VNP(1–36) with more than 99% confidence. Such high sensitivity permitted direct assaying of VNP with only a few microlitres of plasma.

In fresh water eels, the concentration of VNP in the cardiac ventricle was higher than those in the atrium or brain and that of ANP in the ventricle. Thus, VNP seems to be a ventricular hormone. Although ANP is a major circulating hormone in mammals, the plasma concentration of VNP was threefold higher than that of ANP. The RIA coupled with gel-permeation chromatography revealed that a 14 kDa form, probably proVNP, and smaller forms (3–6 kDa) circulate in eel plasma. Reversephase high performance liquid chromatography identified both VNP(1–36) and VNP(1–25) in eel plasma; VNP(1–36) appeared to be a major form.

Journal of Endocrinology (1994) 141, 81–89
Pharmacokinetics and pharmacodynamics of recombinant and urinary human FSH in the male monkey (Macaca fascicularis)
Authors: Weinbauer G F , Simoni M , Hutchison J S , Nieschlag E
Published: December 2008

A dose-finding study was performed in adult male monkeys (Macaca fascicularis) to evaluate the pharmacokinetics and pharmacodynamics of a recombinant human FSH preparation (rhFSH). Groups of five monkeys were randomly assigned to receive single i.m. injections of 0·9% (w/v) NaCl (diluent), 6, 12 or 24 IU rhFSH/kg or 24 IU urinary human FSH/kg (uhFSH). The doses were based on an in vivo ovarian weight gain assay. Blood samples were collected 24 h before and immediately prior to injections, and 4, 8, 12, 24, 72 and 96 h after injections for determination of serum levels of immunoactive FSH by fluoroimmunoassay, bioactive FSH by an in vitro Sertoli cell assay, and inhibin and testosterone by radioimmunoassay. Inhibin was chosen as a marker for in vivo hFSH activity, since the secretion of inhibin in male monkeys is under the control of FSH. Administration of hFSH resulted in dose-related increases in serum hFSH concentrations. rhFSH and uhFSH exhibited similar pharmacokinetics. Comparable findings were obtained when serum samples were analysed for in vitro FSH bioactivity. Maximum serum hFSH levels were obtained 4–6 h after administration and the elimination half-life of hFSH was on average 18–22 h. The serum pharmacokinetics of rhFSH were linear within the dose range explored. Baseline inhibin concentrations varied significantly between groups. However, when the changes in inhibin concentrations were normalized to the baseline values (per cent change, area under curve and maximum inhibin level), a dose-dependent stimulatory effect of rhFSH on serum inhibin was evident. This effect attained statistical significance with doses of 24 IU rhFSH/kg and 24 IU uhFSH/kg, and the serum inhibin responses to rhFSH and uhFSH were not significantly different. No significant differences were observed with regard to the serum concentrations of testosterone between the diluentand hFSH-treated groups. It was concluded that rhFSH is bioactive in terms of stimulating testicular inhibin production in the male monkey and that the pharmacokinetic properties of rhFSH and uhFSH are similar.

Journal of Endocrinology (1994) 141, 113–121