bluuebunny/crossref_metadata_2025 huggingface dataset:
Testosterone-induced inhibition of spermatogenesis is more closely related to suppression of FSH than to testicular androgen levels in the cynomolgus monkey model (Macaca fascicularis)
Authors: Weinbauer GF , Schlatt S , Walter V , Nieschlag E
Published: December 2004

We have investigated the antigonadotropic and antispermatogenic effects of exposure to a long-acting testosterone ester in the cynomolgus monkey model. Groups of five adult animals were exposed either to vehicle or to 10 mg/kg or 20 mg/kg testosterone buciclate (TB) over a 26-week period with injections given in weeks 0, 11 and 18. In week 26, testicular biopsy tissue was collected. Serum testosterone levels were in the upper normal range with 10 mg/kg TB and were approximately twofold higher with 20 mg/kg TB. The estradiol pattern followed that of testosterone and body weights increased in a testosterone-dependent manner. TB completely abolished serum LH bioactivity. Serum concentrations of FSH and inhibin-alpha were suppressed in a TB dose-dependent manner. During weeks 4-8 after the first injection, a rebound of FSH and inhibin but not bioactive LH secretion occurred. This rebound was followed immediately by a restimulation of testis size and sperm numbers. After the next TB injections these parameters were once again suppressed. Nadir testis size was 30-40% of baseline and animals were severely oligozoospermic or transiently azoospermic. Consistent azoospermia was not achieved. Quantitation of serum inhibin B, proliferating cell-nuclear antigen staining and flow cytometric analysis of germ cell populations revealed pronounced suppression of spermatogenesis in both TB-treated groups whereas androgen receptor expression remained unchanged. Testicular androgens levels, determined in week 26, did not differ among all three groups and did not correlate with sperm numbers, histological and immunocytochemical findings. All suppressive effects were fully reversed during the recovery period. We have concluded that pronounced suppression of primate spermatogenesis seemingly requires inhibition of FSH rather than testicular androgen levels, at least in this preclinical non-human primate model. For the purpose of male contraception, FSH inhibition appears mandatory.
Low-dose IGF-I has no selective advantage over insulin in regulating glucose metabolism in hyperglycemic depancreatized dogs
Authors: Fisher SJ , Shi ZQ , Lickley HL , Efendic S , Vranic M , Giacca A
Published: December 2004

At supraphysiological levels, IGF-I bypasses some forms of insulin resistance and has been proposed as a therapeutic agent in the treatment of diabetes. Unfortunately, side effects of high-dose IGF-I (100-250 microg/kg) have precluded its clinical use. Low-dose IGF-I (40-80 microg/kg), however, shows minimal side effects but has not been systematically evaluated. In our previous study under conditions of declining glucose, low-dose IGF-I infusion was more effective in stimulating glucose utilization, but less effective in suppressing glucose production and lipolysis than low-dose insulin. However, under conditions of hyperglycemia, we could not observe any differential effects between high-dose infusions of IGF-I and insulin. To determine whether the differential effects of IGF-I and insulin are dose-related or related to the prevailing glucose level, 3 h glucose clamps were performed in the same animal model as in the previous studies, i.e. the moderately hyperglycemic (175 mg/dl) insulin-infused depancreatized dog, with additional infusions of low-dose IGF-I (67.8 microg/kg, i.e. 29.1 microg/kg bolus plus 0.215 microg/kg( )per min infusion; n=5) or insulin 49.5 mU/kg (9 mU/kg bolus plus 0.45 mU/kg per min; n=7). As in the previous study under conditions of declining glucose, low-dose IGF-I had significant metabolic effects in vivo, in our model of complete absence of endogenous insulin secretion. Glucose production was similarly suppressed with both IGF-I and insulin, by 54+/-3 and 56+/-2% s.e. (P=NS) respectively. Glucose utilization was stimulated to the same extent (IGF-I 5.2+/-0.2, insulin 5.5+/-0.3 mg/kg per min, P=NS). Glucagon, free fatty acid, glycerol, alanine and beta-hydroxybutyrate, were suppressed, while lactate and pyruvate levels were raised, similarly with IGF-I and insulin. We conclude that: (i) differential effects of IGF-I and insulin may be masked under hyperglycemic conditions, independent of the hormone dose; (ii) low-dose IGF-I has no selective advantage over additional insulin in suppressing glucose production and lipolysis, nor in stimulating glucose utilization during hyperglycemia and subbasal insulin infusion when insulin secretion is absent, as in type 1 diabetes mellitus.
Primary hypothyroidism in dogs is associated with elevated GH release
Authors: Lee WM , Diaz-Espineira M , Mol JA , Rijnberk A , Kooistra HS
Published: December 2004

The pulsatile secretion patterns of GH were investigated in seven beagle bitches by collecting blood samples every 10 min for 6 h during euthyroidism and 1.5 years after induction of primary hypothyroidism. Hypothyroidism was induced by surgical removal of the thyroid gland and subsequent destruction of any remnant thyroid tissue by oral administration of sodium [(131)I]iodide. Some of the physical changes observed in the dogs with primary hypothyroidism mimicked those of acromegaly. During both euthyroidism and hypothyroidism GH was secreted in a pulsatile fashion. The mean (+/-s.e.m. ) basal plasma GH concentration was significantly higher (P=0.003) in the hypothyroid state (4.1+/-1.6 microg/l) than in the euthyroid state (1.2+/-0.4 microg/l). Likewise, the mean area under the curve (AUC) for GH above the zero-level during hypothyroidism (27.0+/-10.0 microg/lx6 h) was significantly higher (P=0.004) than that during euthyroidism (11.7+/-2.0 microg/l x 6 h). The mean AUC for GH above the baseline was significantly lower (P=0.008) during hypothyroidism (2.4+/-0.8 microg/l x 6 h) than during euthyroidism (4.5+/-1.8 microg/lx6 h), whereas there was no significant difference in GH pulse frequency. The mean plasma IGF-I level was significantly higher (P<0.01) in the hypothyroid state (169+/-45 microg/l) than in the euthyroid (97+/-15 microg/l). The results of this study demonstrate that primary hypothyroidism in dogs is associated with elevated basal GH secretion and less GH secreted in pulses. This elevated GH secretion has endocrine significance as illustrated by elevated plasma IGF-I levels and some physical changes mimicking acromegaly. It is discussed that the increased GH release in hypothyroid dogs may be the result of the absence of a response element for thyroid hormone within the canine pituitary GH gene and alterations in supra-pituitary regulation.
Regulation of cytochrome P450 aromatase gene expression in adult rat Leydig cells: comparison with estradiol production
Authors: Genissel C , Levallet J , Carreau S
Published: December 2004

Regulation of aromatase gene expression in purified rat Leydig cells has not yet been investigated. Therefore, using a highly specific quantitative RT-PCR method, we have measured the amount of cytochrome P450 aromatase (P450arom) mRNA and aromatase activity in mature rat Leydig cells submitted to various treatments during 24 h. Estradiol production was enhanced in a dose-related manner in the presence of testosterone, the maximum (28% increase) being obtained with 200 ng/ml. Related to the P450arom mRNA levels, a decrease was observed in the presence of low concentrations (50 and 100 ng/ml) of testosterone, then a 20% increase of the amount of transcripts was recorded for the higher concentrations (200-500 ng/ml). The same result was obtained in the presence of 5alpha-dihydrotestosterone (an androgen resistant to aromatase activity). The addition of ovine LH (oLH; 0.1-50 ng/ml) to the Leydig cell culture medium induced a dose-related augmentation of estradiol output up to 10 ng/ml oLH, although a decrease was observed with 50 ng/ml when compared with maximal values. mRNA levels slightly decreased in the presence of low concentrations (0.1-1 ng/ml) of oLH, an effect that was abolished by the addition of testosterone; mRNA levels were increased by oLH (5-10 ng/ml) 35 and 75% respectively in the absence and presence of testosterone (when compared with Leydig cells incubated without treatment). With 50 ng/ml oLH, a large augmentation (twofold) of the P450arom mRNA level either without or with testosterone was observed. Dibutyryl cyclic AMP (1 mM) mimicked the effect of oLH. The half-life of the P450arom mRNAs was twofold increased in the presence of testosterone and oLH when compared with the half-life in the absence of treatment (5.8+/-0.6 h). Taken together, our data have demonstrated that, in freshly isolated Leydig cells from mature rat testes, the regulation of aromatase expression and enzymatic activity is under LH (through cyclic AMP) and steroid control; moreover seminiferous tubule-secreted factor(s) are also involved. Therefore, rat Leydig cell aromatase is controlled at both transcriptional and post-transcriptional steps by endocrine and/or locally produced modulators.
Immunological characterization of human luteinizing hormone with special regard to a common genetic variant
Authors: Nilsson C , Seppala M , Pettersson K
Published: December 2004

Immunoassays are widely used for measuring gonadotropins, including luteinizing hormone (LH), as these are both specific and sensitive. Because LH is microheterogeneous it has been claimed that the specificity of monoclonal antibodies used in two-site immunoassays can limit their clinical utility. Furthermore, we reported earlier a common genetic variant form of LH due to amino acid alterations in the LHbeta gene that is poorly or not recognized by antibodies directed against epitopes present in the intact molecule. We here report the result of an LH epitope mapping using 30 different monoclonal antibodies. The antigenic area affected by the amino acid alterations is fairly large, as antibodies to the two intact domains are able to sandwich each other. Combinations of alpha-beta or beta-beta antibodies generally provide alternatives for unbiased detection of circulating LH and some have reasonably good discrimination of human chorionic gonadotropin. The beta-beta combinations exhibit a peculiarity in urine determinations as they detect the urinary beta-core fragment. Our aims were to study the altered immunoreactivity caused by the amino acid changes and to design two-site LH assays fully capable of recognizing the biologically active LH variant. We also conclude that the variability in recognizing the universally occurring LH variant is the most important factor contributing to the widely documented LH immunoassay discrepancies.
Involvement of beta1- and beta2- but not beta3-adrenoceptor activation in adrenergic PYY secretion from the isolated colon
Authors: Brechet S , Plaisancie P , Dumoulin V , Chayvialle JA , Cuber JC , Claustre J
Published: December 2004

The secretion of PYY by endocrine L cells of the terminal gut is under the control of nutrients, the autonomic nervous system and hormones. Catecholamines, and the non-specific beta-adrenergic agonist isoproterenol induce PYY secretion from rat isolated colon or ileum. Because beta3-adrenergic receptors now appear to mediate many of the effects of catecholamines in the gastrointestinal tract, we investigated the involvement of beta1-, beta2-, and beta3-adrenoceptor stimulation in PYY secretion from the isolated, vascularly perfused rat colon. Infusion of 10(-6) M isoproterenol induced a transient increase in PYY secretion (from 36+/-4 to 87+/-20 fmol/2 min; n=7, P<0.05), that was abolished by a previous infusion of the beta1- and beta2-adrenergic blocker (and partial beta3-agonist) alprenolol (10(-6) M). The beta1-adrenergic agonist dobutamine and the beta-2 agonist terbutaline also (both at 10(-5) M) significantly stimulated PYY secretion, from 29+/-1 to 79+/-12 fmol/2 min and from 19+/-1 to 73+/-13 fmol/2 min respectively (n=7, P<0.05). Neither of the beta3-adrenergic agonists tested (BRL 37 344 (10(-5), 10(-6) M) and SR 58 611A (10(-6) M)) significantly stimulated PYY secretion, thus confirming the exclusive involvement of beta1- and beta2-receptors in beta-adrenergic agonist induced hormone secretion.
Effects of cortisol on chloride cells in the gill epithelium of Japanese eel, Anguilla japonica
Authors: Wong CK , Chan DK
Published: December 2004

The purpose of the present study was to determine the effects of cortisol on the development of the freshwater chloride cell (CC), using flow cytometry. Scanning electron microscopy was used to determine the corresponding modifications in CC apical structure. Simultaneously, biochemical analyses were conducted to determine the activities of transport ATPases, mitochondrial enzymes (succinate dehydrogenase (SDH) and Mg(2+)-ATPase) and lactate dehydrogenase (Ldh). The effects of daily i.m. injection of 2 microg/g cortisol were compared with sham-injected freshwater-, control freshwater- and seawater-adapted fish. The hormone did not affect the activities of Ca(2+)-ATPases in CCs. However, it stimulated the proliferation and differentiation of the two freshwater CC subtypes (F1, 66+/-2.18% (s.e.m. ) and F2, 34+/-2.18%), in which the relative proportion of F1 CCs was transiently reduced in the first 5 days of treatment (F1, 53+/-1.83%; F2, 47+/-1.83%) but was then restored to a higher relative percentage on day 10 (F1, 70+/-1.42%; F2, 30+/-1.42%). Biochemically, it induced the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, SDH and Ldh, suggesting an increase in ion pumping and its associated metabolic activities. CCs from cortisoltreated fish demonstrated recessed apical morphology, accompanied by an increase in cell density (2012 to 2413/mm(2)). Nevertheless, the extent of cell proliferation and differentiation and the biochemical changes were significantly lower than those of seawater fish. Our results indicate that cortisol alone cannot stimulate a complete differentiation of freshwater CCs to seawater CCs. However, the respective roles of the two CC subtypes in freshwater and seawater environments are indicated.
Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats
Authors: Gow RM , O'Bryan MK , Canny BJ , Ooi GT , Hedger MP
Published: December 2004

A single intraperitoneal injection of lipopolysaccharide (LPS) causes a biphasic suppression of testicular steroidogenesis in adult rats, with inhibition at 6 h and 18-24 h after injection. The inhibition of steroidogenesis is independent of the reduction in circulating LH that also occurs after LPS treatment, indicating a direct effect of inflammation at the Leydig cell level. The relative contributions to this inhibition by intratesticular versus systemic responses to inflammation, including the adrenal glucocorticoids, was investigated in this study. Adult male Wistar rats (eight/group) received injections of LPS (0.1 mg/kg i.p.), dexamethasone (DEX; 50 microg/kg i.p.), LPS and DEX, or saline only (controls), and were killed 6 h, 18 h and 72 h later. Treatment with LPS stimulated body temperature and serum corticosterone levels measured 6 h later. Administration of DEX had no effect on body temperature, but suppressed serum corticosterone levels. At the dose used in this study, DEX alone had no effect on serum LH or testosterone at any time-point. Expression of mRNA for interleukin-1beta (IL-1beta), the principal inflammatory cytokine, was increased in both testis and liver of LPS-treated rats. Serum LH and testosterone levels were considerably reduced at 6 h and 18 h after LPS treatment, and had not completely recovered by 72 h. At 6 h after injection, DEX inhibited basal IL-1beta expression and the LPS-induced increase of IL-1beta mRNA levels in the liver, but had no effect on IL-1beta in the testis. The effects of DEX on IL-1beta levels in the liver were no longer evident by 18 h. In LPS-treated rats, DEX caused a significant reversal of the inhibition of serum LH and testosterone at 18 h, although not at 6 h or 72 h. Accordingly, DEX inhibited the systemic inflammatory response, but had no direct effect on either testicular steroidogenesis or intra-testicular inflammation, at the dose employed. These data suggest that the inhibition of Leydig cell steroidogenesis at 6 h after LPS injection, which was not prevented by co-administration of DEX, is most likely due to direct actions of LPS at the testicular level. In contrast, the later Leydig cell inhibition (at 18 h) may be attributable to extra-testicular effects of LPS, such as increased circulating inflammatory mediators or the release of endogenous glucocorticoids, that were inhibited by DEX treatment. These data indicate that the early and late phases of Leydig cell inhibition following LPS administration are due to separate mechanisms.
Prevalence of Gs alpha mutations in Korean patients with pituitary adenomas
Authors: Kim HJ , Kim MS , Park YJ , Kim SW , Park DJ , Park KS , Kim SY , Cho BY , Lee HK , Jung HW , Han DH , Lee HS , Chi JG
Published: December 2004

The reported frequencies of Gs alpha mutations (gsp mutations) in growth hormone (GH)-secreting pituitary adenomas are variable (ranging from 4.4 to 43%), and the presence of these mutations in the other pituitary adenomas is still a matter of controversy. Previous clinical and biochemical analyses of patients with GH-secreting pituitary adenomas and gsp mutations produced conflicting results and did not demonstrate obvious characteristics. Therefore, we investigated the prevalence of gsp mutations in Korean patients with pituitary adenomas and elucidated the characteristics of these patients. Forty-four GH-secreting adenomas, 7 prolactin (PRL)-secreting adenomas and 32 clinically non-functioning adenomas were examined for the presence of point mutations in codon 201 and 227 of the Gs alpha gene using a nested PCR and direct sequencing of DNA extracted from fresh tissue or paraffin-embedded pituitary adenoma samples. Seven of the 44 GH-secreting pituitary adenomas had point mutations at codon 201 or 227; of these, five mutations were in codon 201 and two were in codon 227. In patients with gsp mutations, mean tumor size was significantly smaller than in patients without gsp mutations (15.9+/-8.7 mm vs. 24.9+/-14.9 mm, P<0.05). Age, sex, basal GH levels, GH response to oral glucose loading, GH response to octreotide and surgical outcome were not different in the two groups. One of the 32 clinically non-functioning pituitary adenomas had a point mutation at codon 201; none of the seven prolactinomas had these mutations. These results show that gsp mutations are not rare in Korean acromegalic patients and mean tumor size is significantly smaller in acromegalic patients with gsp mutations. Our results also confirm the low frequency of gsp mutations in clinically non-functioning pituitary adenomas and the absence of gsp mutations in prolactinoma.
Insulin-like growth factors and body growth in chickens divergently selected for high or low growth rate
Authors: Beccavin C , Chevalier B , Cogburn LA , Simon J , Duclos MJ
Published: December 2004

Insulin-like growth factors (IGFs) stimulate growth rate in a number of animal species and are likely to contribute to genetic variations of growth potential. The present study was designed to link levels of IGF-I and IGF-II mRNA and peptides with growth rate in divergently selected genotypes of chickens with high (HG) or low (LG) growth rates. Circulating IGF-I and -II and hepatic mRNA levels were measured under ad libitum feeding conditions from 1 to 12 weeks of age, and at 6 weeks of age under three different nutritional conditions (fed, fasted for 16 or 48 h, re-fed for 4 or 24 h after a 48-h fast). IGF binding proteins (IGFBPs) were also measured. Circulating IGFs increased with age and were higher in HG chickens from 1 to 6 weeks. They decreased with fasting and only IGF-II was fully restored after 24 h of re-feeding, while IGF-I remained low. A significant decrease in steady state IGF-I mRNA levels was also observed with fasting. Across the nutritional study, hepatic IGF-I mRNAs were significantly higher in HG chickens. Variations of IGF-II mRNA levels with nutritional state or genotype exhibited a similar trend. IGFBP (28, 34 and 40 kDa) levels increased with age, while only faint differences were observed between genotypes. IGFBP-28 transiently increased with fasting and was inversely related to blood glucose and insulin levels, suggesting that it is equivalent to mammalian IGFBP-1. In HG chickens, IGFBP-28 and IGFBP-34 levels decreased markedly following re-feeding. Therefore, high and low growth rates were respectively associated with high and low IGF-I and -II levels, supporting the hypothesis of a stimulatory role for both IGFs during post-hatching growth of chickens.
Characterisation of insulin-like growth factor-binding protein-3 binding to a novel receptor on human platelet membranes
Authors: Taylor VL , Spencer EM
Published: December 2004

Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at least 4.6 nM (125)I-labelled IGFBP-3 to occupy all binding sites present on 100 microg of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites for IGFBP-3 on platelets, with a K(d) between 2.6x10(-10) and 8.0x10(-10) M and 1.51-4.89x10(11) binding sites/mg of platelet membrane. Kinetic analysis of (125)I-IGFBP-3 binding to platelet membranes demonstrated a forward rate (k(on)) of 8.1x 10(8) per M per min. The reverse rate constant (k(off)) was calculated to be 1.6x10(-1) per min (t(1/2)=4.2 min) and confirmed experimentally to be 3.3x10(-1) per min (t(1/2)=2.1 min). Binding of (125)I-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with (125)I-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in (125)I-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.
cfos and cjun antisense oligonucleotides block mitogenesis triggered by fibroblast growth factor-2 and ACTH in mouse Y1 adrenocortical cells
Authors: Lotfi CF , Armelin HA
Published: December 2004

In G(0)/G(1) cell cycle-arrested mouse Y1 adrenocortical cells, short pulses (30 min to 2 h) of fibroblast growth factor-2 (FGF2) (5 pM to 1 nM) caused induction of cFos protein by 2 h and onset of DNA synthesis stimulation by 8-9 h. FGF2 dose-response curves for cFos induction (percent labeled nuclei with a specific anti-cFos antibody) and DNA synthesis stimulation (bromodeoxyuridine labeling index) were linearly correlated with a correlation coefficient of 0.969. Inhibition of cFos and cJun protein induction with antisense oligodeoxynucleotides (ODNs) to cfos and cjun mRNAs blocked DNA synthesis stimulation by FGF2. Pulses (up to 2 h) of synthetic ACTH(39) (1 pM to 1 nM) and natural porcine corticotropin A (10 pg/ml to 1 microg/ml) also induced cFos protein and DNA synthesis in G(0)/G(1)-arrested Y1 adrenal cells. ACTH dose-response curves for cFos induction and DNA synthesis stimulation were not correlated. But cfos and/or cjun antisense ODNs blocked DNA synthesis stimulation by ACTH. Thus, signals initiated in FGF2 and ACTH receptors appear to converge to the induction of cfos and cjun genes to trigger DNA synthesis stimulation.
Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice
Authors: Toda K , Okada T , Takeda K , Akira S , Saibara T , Shiraishi M , Onishi S , Shizuta Y
Published: December 2004

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.
Reconstituted normal human breast in nude mice: effect of host pregnancy environment and human chorionic gonadotropin on proliferation
Authors: Popnikolov N , Yang J , Liu A , Guzman R , Nandi S
Published: December 2004

The proliferation of normal human breast epithelial cells in women is highest during the first trimester of pregnancy. In an attempt to analyze this hormonal environment in a model system, the effect of host mouse pregnancy and the administration of human chorionic gonadotropin (hCG) were assessed in normal human breast epithelial cells transplanted into athymic nude mice. Human breast epithelial cells, dissociated from reduction mammoplasty specimens and embedded inside the extracellular matrices comprised of collagen gel and Matrigel, were transplanted into nude mice. Proliferation was measured in vivo by BrdU labeling followed by immunostaining of sections from recovered gels in response to an altered hormonal environment of the host animal. The host animal was mated to undergo pregnancy and the complex hormonal environment of the host animal pregnancy stimulated growth of transplanted human cells. This effect increased with progression of pregnancy and reached the maximum during late pregnancy prior to parturition. In order to determine whether additional stimulation could be achieved, the transplanted human cells were exposed to a second cycle of host mouse pregnancy by immediately mating the animal after parturition. This additional exposure of host mouse pregnancy did not result in further increase of proliferation. The effect of hCG administration on transplanted human cells was also tested, since hCG level is highest during the first trimester of human pregnancy and coincides with the maximal breast cell proliferation. Administration of hCG alone stimulated proliferation of human cells in a dose-dependent manner, and could further enhance stimulation achieved with estrogen. The host mouse mammary gland also responded to hCG treatment resulting in increased branching and lobulo-alveolar development. However, the hCG effect on both human and mouse cells was dependent on intact ovary since the stimulation did not occur in ovariectomized animals. Although hCG receptor transcripts were detected in human breast epithelial cells, raising the possibility of a direct mitogenic action, the hCG effect observed in this study may have been mediated via the ovary by increased secretion of ovarian steroids. In summary, using our in vivo nude mice system, the proliferation of normal human breast epithelial cells could be stimulated by host mouse pregnancy and by administration of hCG.
Endocrine regulation of G-protein subunit production in an animal model of type 2 diabetes mellitus
Authors: Rodgers BD , Bernier M , Levine MA
Published: December 2004

Adipocyte beta-adrenergic sensitivity is compromised in animal models of obesity and type 2 diabetes. Although changes in the membrane concentrations of G-protein alpha subunits (Galpha) have been implicated, it remains to be determined how these changes are affected by insulin resistance in the different animal models. Because previous studies used young animals, we measured the concentrations of Galpha and Gbeta subunits in epididymal fat from aged (48 weeks old) db/db mice and from their lean littermates to more closely reproduce the model of type 2 diabetes mellitus. Levels of immunoreactive Galphas, Galphai(1/2), Galphao and Galphaq/11 were all significantly greater in adipocyte membranes from the db/db mice than in membranes from their lean non-diabetic littermate controls. Levels of Galphai(1) and Galphai(2) were also individually determined and although they appeared to be slightly higher in db/db membranes, these differences were not significant. Although the levels of both Galphas isoforms were elevated, levels of the 42 and 46 kDa proteins rose by approximately 42% and 20% respectively, indicating differential protein processing of Galphas. By contrast, levels of Galphai3 were similar in the two groups. The levels of common Gbeta and Gbeta2 were also elevated in db/db mice, whereas Gbeta1 and Gbeta4 levels were not different. To determine whether these changes were due to insulin resistance per se or to elevated glucocorticoid production, G-protein subunit levels were quantified in whole cell lysates from 3T3-L1 adipocytes that were stimulated with different concentrations of either insulin or corticosterone. Although none of the subunit levels was affected by insulin, the levels of both Galphas isoforms were increased equally by corticosterone in a concentration-dependent manner. Since glucocorticoids are known regulators of Galphas gene expression in many cell types and in adipocytes from diabetic rodents, the results presented herein appear to more accurately reflect diabetic pathophysiology than do those of previous studies which report a decrease in Galphas levels. Taken together, these results indicate that most of the selective changes in G-protein subunit production in adipocytes from this animal model of type 2 diabetes may not be due to diminished insulin sensitivity, but may be due to other endocrine or metabolic abnormalities associated with the diabetic phenotype.
Identification of avian alpha-melanocyte-stimulating hormone in the eye: temporal and spatial regulation of expression in the developing chicken
Authors: Teshigawara K , Takahashi S , Boswell T , Li Q , Tanaka S , Takeuchi S
Published: December 2004

The presence and possible physiological roles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the peripheral tissues of birds have not been established. By a combination of RT-PCR, immunocytochemistry and in situ hybridization, we have examined alpha-MSH expression in the eye of the chicken during development. In the 1-day-old chick, alpha-MSH was expressed in the retinal pigment epithelial (RPE) cells, and also at a lower level in the cone cells. The melanocortin receptor subtypes, CMC1, CMC4 and CMC5, were expressed in the layers of the choroid and the neural retina, but not in the RPE cells. It is probable that the RPE cells secrete alpha-MSH to exert paracrine effects on the choroid and neural retina. During embryonic development, alpha-MSH immunoreactivity in the RPE cells was initially detected at embryonic day 10, and increased in intensity as development proceeded. No cone cells were stained with anti-alpha-MSH antiserum in any of the embryonic stages tested. The immunoreactivities for two prohormone convertases, PC1 and PC2, were co-localized to the RPE cells with a pattern of staining similar to that of alpha-MSH. Despite containing alpha-MSH immunoreactivity, the RPE cells in 1-day-old chicks expressed no immunoreactivity for the endoproteases. Furthermore, in a 3-day-old chick, pro-opiomelanocortin mRNA was detectable by in situ hybridization only in the photoreceptor layer and not in the RPE cells. These results suggest that the RPE cells and the cone cells are intraocular sources of alpha-MSH in the embryonic and postnatal life of the chicken respectively. Embryonic expression of alpha-MSH in the RPE cells implies a possible role for the peptide in ocular development.
The regulation by ovarian steroids of prostaglandin synthesis and prostaglandin-induced contractility in non-pregnant rat myometrium. Modulating effects of isoproterenol
Authors: Engstrom T
Published: December 2004

The objectives of the present study were to investigate the effects of the reproductive steroids oestradiol and progesterone on myometrial levels of cyclooxygenase-2 (COX-2) mRNA and PGF(2alpha) induced myometrial contractility and to study whether the effect of beta(2)-adrenoceptor stimulation by isoproterenol on the myometrium alters these parameters. Oestrogen treatment of ovariectomized rats increased myometrial COX-2 mRNA whereas PGF(2alpha) receptor (PGF(2alpha)-R) mRNA was unchanged following this treatment and maximal contractility (E(max)) of isolated uterine strips challenged with PGF(2alpha) was unaltered. Progesterone treatment alone decreased COX-2 mRNA in comparison with values obtained from oestrogen-treated animals, and in combination with oestrogen the enhancing effect of progesterone on COX-2 mRNA was curbed. EC(50) of uterine strips challenged with PGF(2alpha) increased following oestrogen treatment whereas this parameter was substantially decreased following progesterone treatment. When oestrogen was combined with isoproterenol infusion mRNA values of both COX-2 and PGF(2alpha)-R were reduced. Finally, when isoproterenol infusions were given in combination with both oestrogen and progesterone, PGF(2alpha)-R mRNA and E(max )were enhanced as compared with similar rats not having received isoproterenol. We conclude that oestrogen increases COX-2 mRNA production and subsequent prostaglandin synthesis in non-pregnant rat myometrium. We further conclude that in the oestrogen-dominated rat myometrium the relaxing effect of beta(2)-adrenoceptor stimulation involves attenuation of both prostaglandin synthesis and PGF(2alpha)-R expression. We finally conclude that in the presence of both oestrogen and progesterone this effect of beta(2)-adrenoceptor stimulation is restrained.
Expression and regulation of osteopontin and connective tissue growth factor transcripts in rat anterior pituitary
Authors: Ehrchen J , Heuer H , Sigmund R , Schafer MK , Bauer K
Published: December 2004

Cell-cell interactions are important regulatory elements in anterior pituitary (AP) physiology. As model systems to study pituitary cell-cell interactions, AP cells kept either as monolayers or as organotypic reaggregate cultures were analyzed by differential display PCR. We identified six cDNA fragments (osteopontin (Opn), connective tissue growth factor (CTGF), alpha(v)-integrin, cathepsin H, lysozyme and O-acetyl GD(3) ganglioside synthase) that showed elevated expression in monolayers compared with reaggregate cultures and the AP. The adenohypophyseal mRNA expression of Opn and CTGF, two secreted signaling substances, was studied in more detail. In situ hybridization histochemistry revealed that Opn mRNA expression is restricted to a subpopulation of gonadotropes whereas CTGF hybridization signals could not be ascribed to any known cell type. Opn transcript levels were downregulated in the APs of lactating rats and decreased when rats received s.c. injections of 17beta-estradiol for 5 days. The mRNA expression was higher in male than in female rats and increased after gonadectomy. CTGF transcript levels were higher in male compared with female rats and were increased in pregnant rats and in rats treated for 5 days with triiodothyronine or dexamethasone. These results indicate that Opn and CTGF may be of physiological importance as local communication factors in the AP.
Differential responsiveness of intestinal epithelial cells to 1,25-dihydroxyvitamin D3--role of protein kinase C
Authors: Armbrecht HJ , Boltz MA , Hodam TL , Kumar VB
Published: December 2004

Non-transformed rat intestinal epithelial cell (IEC) lines were used to study the action of 1,25-dihydroxyvitamin D(3) (1,25(OH)2D) in the intestine. The capacity of 1,25(OH)2D to increase the expression of the cytochrome P450 component of the vitamin D 24-hydroxylase (CYP24) was determined in IEC-6 and IEC-18 cell lines. In IEC-6 cells, which are derived from crypt cells isolated from the whole small intestine, 1,25(OH)2D markedly increased expression of CYP24 protein and mRNA within 12 h. In contrast, in IEC-18 cells, which are derived from crypt cells from the ileum only, 1,25(OH)2D did not increase expression of CYP24 until 24-48 h. The maximal levels of CYP24 mRNA seen in the IEC-18 cells were only 31% of the maximal levels seen in the IEC-6 cells. In the presence of 1,25(OH)2D, phorbol esters rapidly increased CYP24 mRNA levels in IEC-18 cells from almost undetectable to levels seen in IEC-6 cells. Protein kinase inhibitors abolished the stimulation by 1,25(OH)2D and by phorbol esters in both cell lines. Stimulation of mRNA levels by phorbol esters required new protein synthesis but stimulation by 1,25(OH)2D did not. These studies demonstrated that the rapid action of 1,25(OH)2D in IEC-6 cells is related to the activation of protein kinase C, an event which is missing in the IEC-18 cells. This differential response to 1,25(OH)2D probably takes place at a post-receptor site, since the number of vitamin D receptors in each cell line was found to be similar.
Endomorphins and activation of the hypothalamo-pituitary-adrenal axis
Authors: Coventry TL , Jessop DS , Finn DP , Crabb MD , Kinoshita H , Harbuz MS
Published: December 2004

Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides recently located in the central nervous system and immune tissues with high selectivity and affinity for the mu-opioid receptor. Intracerebroventricular (i.c.v.) administration of morphine stimulates the hypothalamo-pituitary-adrenal (HPA) axis. The present study investigated the effect of centrally administered EM-1 and EM-2 on HPA axis activation. Rats received a single i.c.v. injection of either EM-1 (0.1, 1.0, 10 microg), EM-2 (10 microg), morphine (10 microg), or vehicle (0.9% saline). Blood samples for plasma corticosterone determinations were taken immediately prior to i.c.v. administration and at various time points up to 4 h post-injection. Trunk blood, brains and pituitaries were collected at 4 h. Intracerebroventricular morphine increased plasma corticosterone levels within 30 min, whereas EM-1 and EM-2 were without effect. In addition, pre-treatment of i.c.v. EM-1 did not block the rise in corticosterone after morphine. Corticotrophin-releasing factor (CRF) mRNA and arginine vasopressin (AVP) mRNA in the paraventricular nucleus (PVN) and POMC mRNA in the anterior pituitary were found to be unaffected by either morphine or endomorphins. Since release of other opioids are elevated in response to acute stress, we exposed rats to a range of stressors to determine whether plasma EM-1 and EM-2 can be stimulated by HPA axis activation. Plasma corticosterone, ACTH and beta-endorphin were elevated following acute restraint stress, but concentrations of plasma EM-1-immunoreactivity (ir) and EM-2-ir did not change significantly. Corticosterone, ACTH and beta-endorphin were further elevated in adjuvant-induced arthritis (AA) rats by a single injection of lipopolysaccharide (LPS), but not by restraint stress. In conclusion, neither EM-1 or EM-2 appear to influence the regulation of the HPA axis. These data suggest that endomorphins may be acting on a different subset of the mu-opioid receptor than morphine. The failure to induce changes in plasma EM-ir in response to the chronic inflammatory stress of AA, the acute immunological stress of LPS, or the psychological stress of restraint, argues against an important role for endomorphins in mediating HPA axis activity.
Cholecystokinin expression in the developing and regenerating pancreas and intestine
Authors: Liu G , Pakala SV , Gu D , Krahl T , Mocnik L , Sarvetnick N
Published: December 2004

In developmental terms, the endocrine system of neither the gut nor the pancreatic islets has been characterized fully. Little is known about the involvement of cholecystokinin (CCK), a gut hormone, involved in regulating the secretion of pancreatic hormones, and pancreatic growth. Here, we tracked CCK-expressing cells in the intestines and pancreata of normal mice (BALB/c), Non Obese Diabetic (NOD) mice and interferon (IFN)-gamma transgenic mice, which exhibit pancreatic regeneration, during embryonic development, the postnatal period and adulthood. We also questioned whether IFN-gamma influences the expression of CCK. The results from embryonic day 16 showed that all three strains had CCK in the acinar region of pancreata, and specifically in alpha cells that also expressed glucagon. However, in adulthood only BALB/c and NOD mice continued this pattern. By contrast, in IFN-gamma transgenic mice, CCK expression was suppressed from birth to 3 months of age in the pancreata but not intestines. However, by 5 months of age, CCK expression appeared in the regenerating pancreatic ductal region of IFN-gamma transgenic mice. In the intestine, CCK expression persisted from fetus to adulthood and was not influenced by IFN-gamma. Intestinal cells expressing CCK did not co-express glucagon, suggesting that these cells are phenotypically distinct from CCK-expressing cells in the pancreatic islets, and the effect of IFN-gamma on CCK varies depending upon the cytokine's specific microenvironment.
Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio)
Authors: van Den Burg EH ,   Metz, Arends RJ , Devreese B , Vandenberghe I , Van Beeumen J , Wendelaar Bonga SE , Flik G
Published: December 2004

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.
GH gene expression in the submaxillary gland in normal and Ames dwarf mice
Authors: Perez-Romero A , Dialynas E , Salame F , Amores A , Vidarte L , Bartke A , Ariznavarreta C , Tresguerres JA
Published: December 2004

High local GH-releasing hormone (GHRH) levels are capable of inducing transdifferentiation in salivary cells to synthesize GH. However, the factors implicated in this process remain unknown. To study this subject, normal and Ames dwarf mice were implanted in the submaxillary gland with a slow release pellet releasing 21 microgram GHRH (1-29)-NH(2)/day for 2 months. Control animals received placebo pellets at the same site. After 60 days, heart blood was collected and submaxillary glands were removed. Circulating levels of GH and IGF-I were significantly decreased (P<0.05) in dwarf mice in comparison with controls, and GHRH treatment did not modify either of these two parameters. Controls carrying GHRH pellets showed a significantly higher GH content (P<0.05) in the submaxillary gland than the placebo-treated normal mice. There were no differences between the IGF-I concentrations of placebo- and GHRH-treated salivary tissue from normal mice. Analysis of GH mRNA by RT-PCR followed by Southern blot revealed that GH transcripts were present in the salivary gland samples carrying the placebo pellets in both normal and dwarf mice. The expression of GH was significantly (P<0.05) increased by the GHRH pellets in salivary tissue from normal mice, but not in submaxillary glands from dwarf mice. Pit-1 mRNA was not detected in the GHRH-treated glands of normal and dwarf mice by RT-PCR or by Southern blot. Using these highly sensitive methods, we have been able to detect the transcription of both GH and Pit-1 in pituitaries from Pit-1-deficient Ames dwarf mice. The present experiment demonstrates that salivary tissue synthesizes GH when it is exposed to the influence of GHRH. Both basal and GHRH-induced salivary GH expression appear to be independent of Pit-1.