bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct infusion of a variant of insulin-like growth factor-I into the skin of sheep and effects on local blood flow, amino acid utilization and cell replication
Authors: Harris P. M. , McBride B. W. , Gurnsey M. P. , Sinclair B. R. , Lee J.
Published: December 2008

In vivo effects of local infusion of a variant of insulin-like growth factor-I (IGF-I), long-R3-IGF-I, into the skin were investigated using six conscious sheep with food available ad libitum. An artery and vein on the abdominal flank of each animal, as well as the saphenous artery, were catheterized so that infusion of isotopically labelled amino acids, with or without IGF-I, could be used to determine amino acid uptake by arteriovenous difference in combination with blood flow determined by dye dilution. Measurements were made on each animal prior to IGF-I infusion, at hourly intervals for the 4 h of IGF-I infusion into the skin artery, then 2 and 4 h after IGF-I infusion ceased. Numbers of cells replicating in the bulbs of wool follicles in the IGF-I-infused area and in the skin on the contralateral side of each animal were measured after labelling with 5-bromo-2′-deoxyuridine. IGF-I caused a significant increase in the skin blood flow (P<0·05), utilization of oxygen (P<0·05), uptake of cysteine (P<0·05) and phenylalanine (P<0·001), and the rate of utilization of cysteine (P<0·05) for protein synthesis. IGF-I increased amino acid uptake regardless of whether the skin was in negative or positive amino acid balance prior to infusion. During the recovery period amino acid utilization by skin returned towards preinfusion levels. No effects of IGF-I were found on replicating cell numbers in the bulbs of wool follicles.

Journal of Endocrinology (1993) 139, 463–472
Immunohistochemical localization of 17α-hydroxylase/C17-20 lyase and aromatase cytochrome P-450 in polycystic human ovaries
Authors: Tamura T. , Kitawaki J. , Yamamoto T. , Osawa Y. , Kominami S. , Takemorit S. , Okada H.
Published: December 2008

Immunohistochemical localization of 17α-hydroxylase/C17-20 lyase (P-45017α,lyase) and aromatase cytochrome P-450 (P-450arom) in polycystic ovary (PCO) syndrome was studied using specific polyclonal antibodies which had been raised against the corresponding enzymes. In the majority of follicles that were atretic and smaller than 7 mm in diameter, theca interna cells showed high P-45017α,lyase immunoreaction, while small numbers of granulosa cells showed little P-450arom immunoreaction. In some atretic follicles that were larger than 11 mm in diameter, the hyperplastic theca interna cell layer showed high immunoreaction to P-45017α,lyase, while the poorly proliferated granulosa cell layer showed a mixture of weak and negative immunoreaction to P-450arom. No immunoreaction to P-45017α,lyase or P-450arom was recognized in PCO stroma. These findings suggest that the theca interna cells and the granulosa cells from PCOs show abnormal steroidogenic function, while the localization of P-45017α,lyase and P-450arom in PCOs was essentially identical to that in the normal ovary. Theca interna cells in PCO atretic follicles are the main site of excess androgen production.

Journal of Endocrinology (1993) 139, 503–509
Circulating insulin-like growth factors-I and -II and substrates in fetal sheep following restriction of placental growth
Authors: Owens J A , Kind K L , Carbone F , Robinson J S , Owens P C
Published: December 2008

To determine the relationship between placental delivery of oxygen and glucose, circulating insulin-like growth factors (IGFs) and fetal growth, the effect of variable restriction of placental growth was determined in sheep in late gestation. Arterial blood was obtained via indwelling catheters at 120 and 127 days of gestation, prior to necropsy at 130 days to measure fetal and placental weights. Plasma was acidified and subjected to size-exclusion high-performance liquid chromatography at pH 2·8 to dissociate and separate IGFs from their binding proteins. The acid-dissociated IGF fraction was analysed by sensitive and highly specific radioligand assays for IGF-I and IGF-II, previously defined using ovine IGFs. Fetal weight and blood pO2 and glucose at 120 and 127 days of gestation correlated positively with placental weight. Plasma IGF-I was positively associated with fetal weight and fetal liver weight, and with blood pO2 and glucose at both ages. Plasma IGF-II levels also correlated positively with fetal weight, fetal liver weight and with blood glucose and pO2, but only at 127 days of gestation. In the most severely growth-retarded fetal sheep, blood glucose and pO2 and plasma IGF-I were significantly reduced when compared with normal fetuses at 120 days. All decreased further by 127 days of gestation as did plasma IGF-II in severely growth-retarded fetal sheep compared with normal fetuses. These observations are consistent with the hypothesis that both IGF-I and IGF-II are chronically regulated by oxygen and nutrition in utero and mediate part of the influence of placental supply of substrate over fetal growth.

Journal of Endocrinology (1994) 140, 5–13
Stimulation of mammogenesis and lactogenesis by recombinant bovine placental lactogen in steroid-primed dairy heifers
Authors: Byatt J C , Eppard P J , Veenhuizen J J , Curran T L , Curran D F , McGrath M F , Collier R J
Published: December 2008

A model of induced lactation was modified to examine the effects of bovine prolactin (bPRL) and bovine placental lactogen (bPL) on mammary growth and differentiation. Thirty-two peripubertal, non-pregnant Holstein heifers were given daily s.c. injections of oestradiol (0·05 mg/kg) and progesterone (0·25 mg/kg) for 7 days to initiate mammary growth. Treatment with bromocriptine (40 mg/3 days) reduced serum PRL concentrations to approximately 25% of pretreatment levels, for the duration of the study. On the day following the last steroid injection, groups of eight heifers were given twice daily s.c. injections of either saline (negative control), recombinant bPRL (rbPRL; 80 mg/day) or recombinant bPL (rbPL; 80 and 160 mg/day) for 7 days. At the end of this period (day 15), growth and differentiation of the mammary glands were assessed. Treatment with rbPL increased total mammary DNA above control value by 50 and 60% for the 80 and 160 mg/day doses respectively. However, total DNA was not different for the control and rbPRL-treated groups. The blood serum concentration of α-lactalbumin was measured daily throughout the study and used as an index of mammary differentiation. Both rbPRL and rbPL stimulated mammary differentiation (i.e. induction of milk synthesis), although rbPRL appeared to be more potent than rbPL. These results indicate that rbPL is lactogenic in vivo and strongly suggest that bPL is a mammary mitogen.

Journal of Endocrinology (1994) 140, 33–43
Structure–activity relationships of glucagon-like peptide-1(7–36)amide: insulinotropic activities in perfused rat pancreases, and receptor binding and cyclic AMP production in RINm5F cells
Authors: Watanabe Y , Kawai K , Ohashi S , Yokota C , Suzuki S , Yamashita K
Published: December 2008

To examine the structure–activity relationships in the insulinotropic activity of glucagon-like peptide-1(7–36) amide (GLP-1(7–36)amide), we synthesized 16 analogues, including eight which were designed by amino acid substitutions at positions 10 (Ala10), 15 (Serl5), 16 (Tyr16), 17 (Arg17), 18 (Lys18), 21 (Gly21), 27 (Lys27) and 31 (Asp31) of GLP-1(7–36)amide with an amino acid of GH-releasing factor possessing only slight insulinotropic activity, and three tentative antagonists including [Glu15]-GLP-1(8–36)amide. Their insulinotropic activities were assessed by rat pancreas perfusion experiments, and binding affinity to GLP-1 receptors and stimulation of cyclic AMP (cAMP) production were evaluated using cultured RINm5F cells.

Insulinotropic activity was estimated as GLP-1(7–36)amide = Tyr16>Lys18, Lys27>Gly21>Asp31⪢Ser15,Arg17>Ala10⪢GRF>[Glu15]-GLP-1(8–36) amide. Displacement activity against 125I-labelled GLP-1 (7–36)amide binding and stimulatory activity for cAMP production in RINm5F cells correlated well with their insulinotropic activity in perfused rat pancreases.

These results demonstrate that (1) positions 10 (glycine), 15 (aspartic acid) and 17 (serine) in the amino acid sequence of GLP-1(7–36)amide, in addition to the N-terminal histidine, are essential for its insulinotropic activity through its binding to the receptor, (2) the amino acid sequences for the C-terminal half of GLP-1(7–36)amide also contribute to its binding to the receptor, although they are less important compared with those of the N-terminal half, and (3) [Glu15]-GLP-1(8–36)amide is not an antagonist of GLP-1(7–36)amide as opposed to des-His1 [Glu9]-glucagon amide which is a potent glucagon antagonist.

Journal of Endocrinology (1994) 140, 45–52
Control of superoxide dismutase, catalase and glutathione peroxidase activities in rat lymphoid organs by thyroid hormones
Authors: Pereira B , Costa Rosa L F B P , Safi D A , Bechara E J H , Curi R
Published: December 2008

This study examined the effect of experimental hyperand hypothyroidism on the superoxide dismutase, catalase and glutathione peroxidase activities of rat lymphoid organs (mesenteric lymph nodes, spleen and thymus) and muscles (soleus and gastrocnemius-white portion) for comparison. The capacity for the generation of reducing equivalents was also investigated: activities of glucose-6-phosphate dehydrogenase (pentose-phosphate pathway) and citrate synthase (Krebs cycle). Hyperthyroidism tended to enhance lipid peroxide content in all tissues. This effect may result from (1) a high capacity for the generation of reducing equivalents in cytosol and mitochondria and (2) a reduced activity of catalase in the lymphoid organs and of glutathione peroxidase in the muscles. The process of lipid peroxidation in these tissues caused by hyperthyroidism was probably slowed down by the augmentation of CuZn- and Mn-superoxide dismutase (Mn-SOD) activities observed under this condition. Hypothyroidism tended to diminish lipid peroxidation and did not affect citrate synthase and glucose-6-phosphate dehydrogenase activities in the lymphoid organs and muscles. Low levels of thyroid hormones tended to diminish Mn-SOD and glutathione peroxidase activities. These findings show that the thyroid hormones might be able to regulate the activities of CuZn- and Mn-SOD, and catalase and glutathione peroxidase in the lymphoid organs and skeletal muscles.

Journal of Endocrinology (1994) 140, 73–77
Changes in fetal thyroid hormone levels in adrenalectomized fetal sheep following continuous cortisol infusion 72 h before delivery
Authors: Sensky P L , Roy C H , Barnes R J , Heath M F
Published: December 2008

Continuous infusion of cortisol into adrenalectomized fetal sheep during the last 72 h of gestation (term=145 ± 2 days) produced a significant (P<0·05) rise in fetal serum tri-iodothyronine (T3) mean ± s.e.m. concentrations from 398 ± 65 to 1340 ± 238 ng/l. A concurrent decrease in plasma thyroxine (T4) levels was observed in three out of four animals. No significant changes in the concentrations of either hormone were noted prior to the start of cortisol infusion. The plasma concentrations of cortisol, T3 and T4 at term were similar to those in untreated full-term lambs. Adrenalectomized fetuses not given cortisol infusions still had low levels of T3 at term, with no increase being observed. The results suggest that cortisol plays an important role in the increase of fetal plasma T3 observed towards the end of gestation. This is probably achieved by the stimulation of the monodeiodination of T4 to T3 in the peripheral tissues.

Journal of Endocrinology (1994) 140, 79–83
Comparison of extraction methods for insulin-like growth factor-binding proteins prior to measurement of insulin-like growth factor-I in undernourished neonatal and adult rat serum
Authors: Rivero F , Goya L , Pascual-Leone A M
Published: December 2008

Insulin-like growth factors (IGFs) are considered to be endocrine regulators during development, and different species have been used for the study of these factors during the perinatal period. The neonatal rat is a very useful model widely utilized to study endocrine alterations throughout the developmental period; few references in the literature present the neonatal rat as a model for the study of IGFs however.

This study was undertaken to compare two extraction methods, acid–ethanol cryoprecipitation (AEC) and formic acid–acetone (FA), for the removal of IGF-binding proteins (IGFBPs) from neonatal and adult rat serum in fed (control) and undernourished populations prior to measurement of IGF-I by radioimmunoassay (RIA). The IGF-I values obtained by RIA following AEC or FA extraction were compared with those obtained following gel filtration (GF), which is considered to be the reference method. Western ligand blotting was used to determine IGFBPs in unextracted serum and after AEC or FA extraction of serum from rats of 10 and 20 days of age and adult rats in both populations. Although serum IGF-I levels after AEC or FA in adult control rats were comparable with those obtained following GF, a significant correlation was found only after AEC extraction both in fed and undernourished adult rats. During the neonatal period, at 10 and 20 days, serum IGF-I levels after AEC or FA extraction were very different from those obtained after GF, especially in undernourished populations, and the correlation was very poor at 10 and 20 days of age in both populations studied.

The IGFBP radioligand blots showed a different rate of change in control and undernourished rats during the period studied. Changes in IGFBP-3 and the band at 30 kDa are reported in both control and undernourished animals. The different proportions of the three major IGFBPs remaining after AEC or FA extraction were also shown. We conclude that AEC extraction, originally validated for use in human serum, is also satisfactory for use in adult but not in neonatal rat serum. However, FA extraction was not a useful method for the rat serum. In neonatal rat serum, the only reliable extraction method was GF.

Journal of Endocrinology (1994) 140, 257–263
Effects of gonadotrophin-releasing hormone during the pubertal development of the male African catfish (Clarias gariepinus): gonadotrophin and androgen levels in plasma
Authors: Schulz R W , van der Sanden M C A , Bosma P T , Goos H J Th
Published: December 2008

The sensitivity of the pituitary to gonadotrophin-releasing hormone (GnRH) and that of the testis to gonadotrophin (GTH) was monitored in African catfish in vivo at different stages of pubertal development (20, 21, 24, 31, 39, 42 and 49 weeks of age). The fish were injected i.p. with chicken GnRH-II (cGnRH-II) or catfish GnRH (cfGnRH), their two endogenous GnRHs. Blood samples were collected to quantify LH-like GTH-II and three androgens 11-ketotestosterone (11-KT), testosterone and 11β-hydroxyandrostenedione (OHA). The testes of 20- and 21-week-old fish contained spermatogonia alone, or spermatogonia and spermatocytes, or – in a limited number of specimens – some spermatids as well. Spermatozoa were first observed in the testes of 24-week-old fish and became predominant as the fish attained full maturity (49 weeks of age). In 20- to 24-week-old fish, significantly elevated plasma GTH-II levels were only recorded after treatment with cGnRH-II. In 31- to 49-week-old fish, injection of both GnRHs led to increased plasma GTH-II levels, but cGnRH-II was always more effective than cfGnRH. Whereas basal GTH-II plasma levels hardly changed throughout the study, GnRH-stimulated levels increased with the age of the fish. Plasma concentrations of 11-KT were not different from controls in 20- and 21-week-old males despite their elevated GTH-II levels following injection of cGnRH-II. The first significant increase in levels of 11-KT after cGnRH-II treatment was observed in 24-week-old fish and, after cfGnRH treatment, in 39-week-old fish. Basal and GnRH-stimulated 11-KT plasma levels increased with the age of the fish. Basal and cGnRH-II-stimulated plasma levels of OHA and testosterone also increased with the age of the fish. However, the levels of OHA and testosterone were five- to tenfold lower than those of 11-KT and, except for OHA in the 49-week-old fish, no increases were recorded in the cfGnRH-injected fish. Our data show that at the beginning of spermatogenesis the pituitary gland is already sensitive to GnRH stimuli. However, sensitivity of the testicular steroidogenic system to GTH-II, sufficient to be reflected in consistently elevated androgen plasma levels, was not observed until 3–4 weeks later. The restricted testicular GTH-II responsiveness at the beginning of spermatogenesis may represent a limiting factor for further pubertal development.

Journal of Endocrinology (1994) 140, 265–273
Effect of growth hormone on steroidogenesis, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 production and DNA synthesis in cultured human luteinized granulosa cells
Authors: Ovesen P , Ingerslev H J , Ørskov H , Ledet T
Published: December 2008

Numerous clinical and experimental observations have suggested that GH is important in ovarian function. We have investigated the effect of GH alone and GH in combination with FSH on the secretion of oestradiol, progesterone, insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) and on [3H]thymidine incorporation in cultured human luteinized granulosa cells. Granulosa cells from patients undergoing treatment for in vitro fertilization were isolated and cultured for 2 days in culture medium with 10% serum. After this preincubation, the medium was removed and the cells were incubated with GH (1, 10 and 100 μg/l) with or without FSH in serum-free medium and in the presence of [3H]methylthymidine (2 μCi/ml). GH alone resulted in a significant dose-dependent increase of oestradiol (P<0·05) and in IGFBP-1 (P<0·002) in the medium. The release of IGF-I was undetectable and there was no increase in [3H]thymidine incorporation with GH alone. Neither GH nor FSH alone stimulated granulosa cell proliferation or progesterone release, while the combination induced increases (P<0·001) in both. The stimulatory effect of GH on steroidogenesis, IGFBP-1 production and granulosa cell proliferation supports a putative role for GH in the regulation of ovarian function.

Journal of Endocrinology (1994) 140, 313–319
Immunocytochemical identification of a relaxin-like protein in gastrointestinal epithelium and carcinoma: a preliminary report
Authors: Stemmermann G N , Mesiona W , Greenwood F C , Bryant-Greenwood G D
Published: December 2008

Immunostaining with antibodies to human relaxin (H2) suggests the presence of a relaxin-like peptide in the gastrointestinal tract and its tumours. The peptide is present in the cytoplasm of non-neoplastic cells at sites of cell exfoliation: the surface cells of the stomach, the small intestinal villi and the surface cells of the colon. It also occurs in the cytoplasm of gastric parietal cells and carcinomas of both the stomach and the colon. Relaxin has been shown to stimulate collagenase gene expression and to down-modulate collagen synthesis and secretion in human skin fibroblasts. The distribution of relaxin shown here suggests that it could play a role in the detachment of non-neoplastic cells from their basement membranes at the end of the cell cycle and in the penetration of carcinoma cells through the basement membrane.

Journal of Endocrinology (1994) 140, 321–325
Interleukin-1β stimulates cell proliferation in the intermediate lobe of the rat pituitary gland
Authors: Stępień H , Żerek-Mełen G , Mucha S , Winczyk K , Fryczak J
Published: December 2008

Interleukin-1 (IL-1) is a multifunctional monokine which possesses an impressive array of diverse actions relating to the function of the immune system. IL-1 is present and formed locally in the brain as demonstrated by biochemical and immunocytochemical methods. Various immunomodulatory and neuroendocrine effects of IL-1 have been reported, including induction of several morphological changes in the endocrine cells of experimental animals and humans. IL-1 is present in two molecular forms (IL-1α and IL-1β) that activate specific receptors for IL-1. In the present study we investigated the possible effect of recombinant human IL-1α and IL-1β and recently cloned anti-human IL-1 receptor antibody (M10) on cell proliferation in the anterior and the intermediate lobe of the pituitary gland of the rat. In vivo labelling with bromodeoxyuridine (BrdU) and immunocytochemical staining with anti-BrdU monoclonal antibody were used as a sensitive index of cell proliferation. IL-1β was found to stimulate dose-dependently (0·1–10 μg/kg body weight) incorporation of BrdU into pituitary intermediate cell nuclei, and positive correlation between the tested doses of IL-1β and BrdU-labelling index was noted (r=0·89; P<0·01). This IL-1β-induced stimulation of pituitary pars intermedia cell proliferation was receptor specific, since stimulation was blocked by anti-IL-1 receptor antibody. On the other hand, recombinant human IL-1α did not affect BrdU incorporation and the proliferation of pituitary pars intermedia cells. In addition, neither of the cytokines tested in the same experimental conditions showed any effect on the cell growth of the pituitary pars anterior. These results suggest that IL-1β may be involved in the regulation of the cell growth of the pituitary intermediate lobe in rats.

Journal of Endocrinology (1994) 140, 337–341
Influence of thyroid hormone on androgen metabolism in peripuberal rat Sertoli cells
Authors: Panno M L , Beraldi E , Pezzi V , Salerno M , De Luca G , Lanzino M , Le Pera M , Sisci D , Prati M , Palmero S , Bolla E , Fugassa E , Andò S
Published: December 2008

The aim of the present study was to investigate the influence of thyroid hormones on androgen metabolism in Sertoli cells isolated from 3- and 4- week-old rats.

Hypothyroidism was induced by the oral administration of 0·025% methimazole (MMI) from birth until the rats were killed at 3 and 4 weeks of age. Half of the MMI-treated animals were injected i.p. with l-tri-iodothyronine (T3 3 μg/100 g body weight) during the last week before death. Sertoli cells from all groups were initially cultured under basal conditions for the first 24 h and subsequently in the presence of testosterone with or without T3 for an additional 24 h. Hypothyroidism was associated with severe impairment of body as well as testicular growth. Indeed, body and testicular weights were similar in 4-week-old hypothyroid animals to those in 3-week-old control rats.

Testosterone metabolism in Sertoli cells isolated from 3- and 4-week-old hypothyroid rats was mainly expressed by the lowering of 5α-dihydrotestosterone + androstane 3α, 17β–diol and an enhanced formation of 5α-reduced steroids with poor androgenic properties (e.g. 5α–androstane, 3, 17α-dione (androstanedione), 5α–androstan, 3-ol-17-one (androsterone)). Treatment of the same group of animals with T3 in vivo and in vitro did not influence the pattern of 5α–reductase steroids substantially.

The most striking finding in the Sertoli cells of 3-week-old hypothyroid rats was the dramatic enhancement of oestradiol formation which persisted to a lesser extent 1 week later. Oestradiol formation was greatly decreased by the addition of T3 in vivo and in vitro in hypothyroid animals.

These results suggest that T3 might influence androgen metabolism during the functional maturation of Sertoli cells.

Journal of Endocrinology (1994) 140, 349–355
Effects of oestradiol-17β and 5α-dihydrotestosterone on guinea-pig prostate smooth muscle cell proliferation and steroid receptor expression in vitro
Authors: Ricciardelli C , Horsfall D J , Sykes P J , Marshall V R , Tilley W D
Published: December 2008

Smooth muscle cells (SMCs) are the major cellular component of the prostatic stroma. The aim of this study was to examine the effects of oestradiol-17β (OE2) and 5α-dihydrotestosterone (DHT) on the proliferation of guinea-pig prostate SMCs in vitro. OE2 stimulated SMC DNA synthesis at all concentrations examined. At a plating density of 3·0 × 104 cells/cm2, maximal incorporation of [3H]thymidine (136% of control) was observed after 36 h of treatment with 1 nmol OE2/l. At the same plating density, DHT had an inhibitory effect on SMC DNA synthesis, with maximal effects (73% of control) being observed 24 h after treatment with 1 nmol DHT/l. These effects of OE2 and DHT were prevented by co-incubation with specific steroid receptor antagonists. At a threefold lower plating density (1·0 × 104 cells/cm2), the maximal stimulatory and inhibitory effects of OE2 and DHT were delayed by approximately 24 and 12 h respectively. At the lower plating density, a biphasic effect of DHT was observed on DNA synthesis; DHT was both inhibitory and stimulatory. Maximal inhibition (71% of control) and maximal stimulation (168% of control) were observed after 36 and 134 h treatment with DHT respectively. At the lower plating density, longer term treatment of SMC cultures with OE2 and DHT also resulted in an increase in cell number. After 7 days of treatment with OE2 and DHT, cell number increased by 13% and 12% respectively. When OE, and DHT were added in combination, the short-term inhibitory effect of DHT on SMC DNA synthesis was dominant over the stimulatory effect of OE2. Treatment with DHT for 24 h significantly inhibited OE2-induced stimulation of [3H] thymidine incorporation, irrespective of the prior duration of OE2 treatment. At the lower plating density, OE2 also decreased oestrogen receptor (ER) mRNA levels to 38% of control levels after 24 h of treatment. ER mRNA levels remained repressed until 72 h after treatment with OE2, and returned to control values following 96 h of treatment. Both the androgen-induced inhibition and stimulation of DNA synthesis observed following treatment of SMCs with 1 nmol DHT/l were associated with a reduction in androgen receptor (AR) mRNA levels. At an intermediate time (i.e. 48 h after commencement of treatment with DHT) AR mRNA levels were increased more than twofold over control levels. The increase in AR mRNA levels at 48 h after DHT treatment only occurred in cells plated at the lower density, suggesting that this is an essential requirement for the longer term stimulation of prostatic SMC proliferation by DHT.

Journal of Endocrinology (1994) 140, 373–383
Expression of 17β-hydroxysteroid dehydrogenase in the rat ovary during follicular development and luteinization induced with pregnant mare serum gonadotrophin and human chorionic gonadotrophin
Authors: Ghersevich S A , Poutanen M H , Rajaniemi H J , Vihko R K
Published: December 2008

Antibodies against human placental 17β-hydroxysteroid dehydrogenase (17-HSD) and 17-HSD cDNA were used to study the expression of the corresponding enzyme in the immature rat ovary during follicular development and luteinization, which were induced by treating the animals with pregnant mare serum gonadotrophin (PMSG) or with PMSG followed by human chorionic gonadotrophin (hCG). Immuno-blot analysis indicated that the Mr of the 17-HSD expressed in rat granulosa cells was 35 000, as previously shown for the human placental enzyme. In immunohistochemical studies of untreated immature rat ovaries, only the granulosa cells from small antral follicles were stained. One day after PMSG treatment, strong expression of 17-HSD was observed in the granulosa cells of growing Graafian follicles. A marked decrease in enzyme expression was observed in preovulatory follicles on day 2 of PMSG treatment, starting from the basal layers of granulosa cells and progressing toward the luminal cells. No 17-HSD expression was detected in luteinized follicles or corpora lutea 22 h after hCG injection. The stroma and theca cells were negative for 17-HSD staining. In Northern hybridization analyses, two 17-HSD mRNAs were detected (1·4 and 1·7 kb). The strongest expression for both mRNAs was detected after 1 day of PMSG treatment, coinciding with maximal immunostaining of the enzyme protein. Down-regulation of 17-HSD observed by immunohistochemistry was reflected in a similar decrease in mRNA expression and the signals were almost undetectable 22 h after hCG injection. Our data suggest that 17-HSD expression in rat granulosa cells is up-regulated during follicular development and, thereafter, the enzyme expression is down-regulated during luteinization.

Journal of Endocrinology (1994) 140, 409–417
Effect of thyroid hormones and other iodinated compounds on the transition of monocytes into veiled/dendritic cells: role of granulocyte-macrophage colony-stimulating factor, tumour-necrosis factor-α and interleukin-6
Authors: Mooij P , Simons P J , de Haan-Meulman M , de Wit H J , Drexhage H A
Published: December 2008

Stimulation of human peripheral blood monocytes with the thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) enhanced their ability to mature into cytologically and functionally characteristic veiled/dendritic cells. Veiled/dendritic cell transition induced by T3 and T4 was dependent on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) in the culture, since the addition of antibodies specific for GM-CSF, TNFα and IL-6 to the culture system had blocking effects. The addition of antibodies to macrophage colony-stimulating factor and IL-1 had no effects. Contaminating T cells and B cells did not contribute to the transition of monocytes to veiled/dendritic cells, and it is therefore likely that the GM-CSF, TNFα and IL-6 produced in the culture system were derived from the monocytes themselves.

Stimulation of the blood monocytes with an optimal concentration of metrizamide (14·5%), reverse T3 (rT3; 2 × 10−10 m) or highly iodinated thyroglobulin (Tg; 2 × 10−11 m) also resulted in an increased transition of monocytes to veiled/dendritic cells, but to a lesser extent in comparison with the thyroid hormones (T3, 31 ±6% and T4, 25 ±5% vs rT3, 22 ± 8% and Tg with an iodination grade of 0·37%: 20 ± 4% veiled/dendritic cells). Administration of anti-GM-CSF, anti-TNFα and anti-IL-6 to the culture system also had blocking effects on the transition from monocytes to veiled/dendritic cells induced by the iodinated compounds. The mechanisms by which such iodinated compounds act on the monocyte to veiled/dendritic cell transition can only be speculated on (interference H2O2-generating system?).

Journal of Endocrinology (1994) 140, 503–512
The distribution of hypothalamic nitric oxide synthase mRNA in relation to gonadotrophin-releasing hormone neurons
Authors: Grossman A.B. , Rossmanith W.G. , Kabigting E.B. , Cadd G. , Clifton D. , Steiner R.A.
Published: December 2008

Using probes for rat neural nitric oxide synthase (NOS) mRNA and GnRH mRNA, we performed in situ hybridization to survey NOS mRNA distribution within the hypothalamus of the male and female rat and sought evidence for its expression in GnRH neurons. The NOS cRNA probe was radiolabelled with 35S, and a digoxigenin-labeled rat GnRH cRNA probe was used for double-label studies. NOS mRNA was localized in discrete hypothalamic areas, in grain clusters suggestive of individual neurons. NOS mRNA-positive cells were located mainly in the supraoptic and paraventricular nucleus, particularly overlying the magnocellular division. Rostrally, cells expressing NOS mRNA were especially prominent in the diagonal band of Broca, in a distribution very similar to GnRH neurons. Nevertheless, only one of 370 cells labeled for GnRH mRNA appeared to be positive for NOS mRNA. We conclude that NOS mRNA is located prominently in regions where CRH, AVP and oxytocin cells are located. NOS mRNA-positive cells are located in close proximity to GnRH neurons, but rarely do such neurons express NOS mRNA.
ACTIVATION OF MAP KINASE ASSOCIATED WITH THE PRIMING EFFECT OF LHRH
Authors: Mitchell R. , Sim P.J. , Leslie T. , Johnson M.S. , Thomson F.J.
Published: December 2008

A MAP kinase activity assay was developed to determine whether the LHRH receptor could activate this enzyme (particularly during LHRH priming). In anterior pituitary tissue from prooestrous rats LHRH caused concentration-dependent activation of MAP kinase after 5-10 min and continued for up to 60 min of incubation. The magnitude of this response correlated with that of LHRH priming on various days of the oestrous cycle but not with the magnitude of 1st hour (unprimed) LHRH-induced LH release. The response to LHRH was mimicked by a phorbol ester but not by ionomycin and was blocked with high potency by GF 109203X but not by H7 (in a similar manner to the PKC species that mediates LHRH priming). Neither the tyrosine kinase inhibitor lavendustin A nor the protein synthesis inhibitor cycloheximide blocked LHRH-induced MAP kinase activation. The possible functional significance of MAP kinase activation in gonadotrophs is considered with respect to LHRH priming.
Induction of ovulation by endothelin-3 in rats
Authors: Furudate S , Masaki Y , Muto T
Published: December 2008

Endothelins vary in their biological activity. We therefore examined the effects of endothelin-3 (ET-3) on ovulation and secretion of LH, FSH and prolactin in rats in which naturally occurring ovulation was blocked by the administration of sodium pentobarbital (40 mg/kg, i.p.) prior to the critical period (1330 h) on the day of pro-oestrus. ET-3 (10 nmol/kg) was given via the jugular vein under pentobarbital anaesthesia from 1600 to 1800 h on the day of pro-oestrous and induced ovulation in all rats whether given by venous injection or by infusion but the number of ova in rats injected with ET-3 was less than that in normally cycling control rats. Infusion of ET-3 stimulated the secretion of LH but caused a lower than expected rate of secretion of FSH. It would therefore appear that ET-3 causes release of the total amount of LH that is required for induction of ovulation. Our findings strongly suggest that ET-3 has a physiologically significant role in the regulation of anterior pituitary hormone secretion.

Journal of Endocrinology (1994) 141, 109–112
Xenopus laevis oocytes, eggs and tadpoles contain immunoactive insulin
Authors: de Pablo F , Dashner R , Shuldiner A R , Roth J
Published: December 2008

Insulin is a multifunctional polypeptide hormone that regulates metabolic processes and promotes mitogenesis and differentiation in vitro in the cells and tissues of several species. Its role in vivo during embryogenesis is still poorly understood. We have previously found insulin mRNA in mature Xenopus laevis oocytes and in embryos during neurulation (before organogenesis of the pancreas takes place). We have now measured insulin immunoactivity in mature oocytes, unfertilized eggs and day-2 tadpoles. Using reversed phase high performance liquid chromatography, we found low levels of insulin in extracts of oocytes (stage VI). Both Xenopus insulin I and II were detected in unfertilized eggs. The day-2 tadpoles (stages 31–33) also contained immunoactive insulin, and in swimming tadpoles (stage 46) a few clusters of cells containing insulin immunoactivity could be identified by indirect immunofluorescence. Immunoblot analysis was relatively insensitive, detecting insulin only in the adult Xenopus pancreas. In summary, insulin (from maternal origin and embryonic expression) appears to be present early enough in Xenopus laevis to influence developmental processes such as neurulation.

Journal of Endocrinology (1994) 141, 123–129
Expression of inhibin subunit mRNAs and inhibin levels in the testes of rats with stage-synchronized spermatogenesis
Authors: Klaij I A , van Pelt A M M , Timmerman M A , Blok L J , de Rooij D G , de Jong F H
Published: December 2008

Inhibin α- and βB-subunit mRNA expression, and levels of bioactive and immunoreactive inhibin were studied in rat testes, synchronized for the stage of the cycle of the seminiferous epithelium by treating vitamin A-deficient rats with vitamin A. Measurement of inhibin subunit mRNA expression and inhibin levels was started directly after the start of vitamin A treatment, and continued for 65 days.

Inhibin subunit mRNA expression, and testicular bioactive and immunoreactive inhibin levels increased after the start of vitamin A treatment, reaching maximum values after 9 days, when B spermatogonia and preleptotene spermatocytes had appeared in the stage-synchronized testes. The ratio between βB- and α-subunit mRNA expression was high at that time-point, whereas the ratio between bioactive and immunoreactive inhibin remained low. These data suggest a relatively high production of activin at that moment, and this may play a role in the development of B spermatogonia into preleptotene spermatocytes during the initiation of spermatogenesis.

Stage-dependency was demonstrated for inhibin subunit mRNA expression, and for the levels of bioactive and immunoreactive inhibin, in rats with complete spermatogenesis. Inhibin α-subunit mRNA expression was relatively high at stages V and XIII of the spermatogenic cycle, whereas βB-subunit mRNA expression was high at stage XIII but not at stage V. This resulted in a high βB/α subunit mRNA ratio at stage XIII. Since it has been shown that expression of the activin receptor is high at stages XIII–I, locally formed activin might play a role in the regulation of meiosis. Bioactive and immunoreactive inhibin were highly correlated during the cycle, with maximum levels at stages XIV–I.

It was concluded that the production of inhibin, and possibly activin, is dependent on the stage of the cycle of the seminiferous epithelium; these growth factors might play a paracrine role in the differentiation of spermatogenic cells.

Journal of Endocrinology (1994) 141, 131–141
Testicular and serum levels of inhibin and expression of inhibin subunit mRNAs are differentially affected by hemicastration in rats of various ages
Authors: Klaij I A , Timmerman M A , Kramer P , Meijs-Roelofs H M A , de Jong F H
Published: December 2008

Age-related short-term effects of hemicastration on testicular weight, serum FSH, immunoreactive inhibin, LH and testosterone, testicular levels of inhibin subunit mRNA expression, and bioactive and immunoreactive inhibin were studied in rats of 8, 15 and 22 days of age. Hemicastration led to an increased weight of the remaining testis after 24 h in 8- and 15-day-old rats, but not in 22-day-old rats. Serum FSH levels were elevated in all hemicastrated rats after 8 h. However, serum immunoreactive inhibin levels were decreased only after 72 h in 8-day-old rats and after 24 h in 15- and 22-day-old rats. Inhibin α-subunit mRNA expression was increased in the testes of hemicastrated rats of 8 and 15 days of age, whereas inhibin βB-subunit mRNA expression was elevated in the testes of 15-day-old rats but not in those of 8- and 22-day-old rats. The increase in α-subunit mRNA content per testis was caused by an increased concentration and increased testicular weight, whereas the increase in βB-subunit mRNA in the remaining testis parallelled the increased testicular weight, indicating that different mechanisms play a role in the regulation of these mRNAs. In 22-day-old rats, a transiently decreased expression of inhibin βB-subunit mRNA was observed 8 h after hemicastration. The increased inhibin α- and βB-subunit mRNA expression in 8- and 15-day-old rats did not result in increased testicular bioactive and immunoreactive inhibin content of the remaining testis, whereas in 22-dayold rats an increased immunoreactive inhibin content of the remaining testis was observed. These data indicate that efficiency of translation, post-translational modifications or transport from the testis play an important role in determining the final testicular content of inhibin.

In conclusion, the response of the remaining testis and the role of inhibin in the regulation of the pituitary-testis axis after unilateral castration depend on the age at which the animals are hemiorchidectomized.

Journal of Endocrinology (1994) 141, 143–151
The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCGβ-core fragment
Authors: Dirnhofer S , Madersbacher S , Bidart J-M , Kortenaar P B W Ten , Spöttl G , Mann K , Wick G , Berger P
Published: December 2008

The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively.

Journal of Endocrinology (1994) 141, 153–162
ACTH-induced preterm labour in the ewe is associated with increased mRNA and protein levels of myometrial gap junction protein, connexin-43
Authors: McNutt C M , Nicholson B J , Lye S J
Published: December 2008

The myometrial gap junction protein, connexin-43, is thought to be critical to the development of synchronous, high-amplitude contractions of the myometrium during labour. The purpose of this study was to determine the relationship between the temporal expression of myometrial connexin-43 mRNA and protein, the contractile patterns of the uterus and the changes in maternal plasma oestrogen and progesterone. On day 127 chronically catheterized fetal sheep were randomized to receive either ACTH (1 μg over 15 min every 2 h) or saline (control) infusions. Using this model, ACTH induces labour in 110 ± 5 h and produces similar endocrine profiles and changes in myometrial activity to that of term spontaneous labour. Myometrial tissue was obtained during autopsy at: 0 h (127 days: no infusion), 72 h saline, 72 h ACTH, 120 h saline, and during ACTH-induced labour (n=4/group). Northern analysis demonstrated a significant (P<0·05) increase in connexin-43 transcripts during labour (2·21±0·39; mean ±s.e.m. relative to 18S) compared with 0 h (0·67±0·17) and 72 h ACTH 0·41 ±0·11). Connexin-43 protein (as determined by Western analysis) showed a similar pattern to that of the transcripts. These changes in myometrial connexin-43 expression were associated with significant increases in the rate of rise of intrauterine pressure, frequency and maximum amplitude of uterine contractions and the maternal plasma oestrogen to progesterone ratio. No changes in connexin-43 expression, contractile parameters or endocrine profiles occurred in control animals. Our data suggest that the increased myometrial contractile coordination during preterm labour in the sheep is, at least in part, due to a steroid-associated increase in connexin-43 expression.

Journal of Endocrinology (1994) 141, 195–202
Effects of dexamethasone on muscle protein homeostasis and on calpain and calpastatin activities and gene expression in rabbits
Authors: Yeh J-Y , Ou B-R , Forsberg N E
Published: December 2008

The objectives were to investigate the mechanisms by which glucocorticoids control proteolysis in muscle cells and the relationship between the calpain:calpastatin system and proteolysis in muscle. Female rabbits were treated with 1 mg dexamethasone (Dex)/kg body weight per day for 0, 1, 2 or 4 days after which animals were killed and muscle samples taken for analyses. Dex reduced urinary Nτ-methylhistidine (NMH) 48% (day 4 versus day 1 of Dex treatment) and muscle NMH concentrations by 49% (day 1) to 40% (day 2) respectively, suggesting that protein degradation was reduced. To investigate whether the changes in apparent proteolysis were related to calpains, we examined the effects of Dex on muscle calpain and calpastatin activities. These were unaffected by Dex. This implies that Dex-dependent changes in degradation are not mediated by changes in muscle calpain or calpastatin activities. We studied the effects of Dex on calpain and calpastatin gene expression as a means of clarifying the relationships between proteinase gene expression and proteinase activities. μ-Calpain mRNA concentration was unaffected by Dex but m-calpain mRNA and calpastatin mRNA concentrations were reduced by 42–55% and 40% respectively. Dex had a similar effect on β-actin mRNA. Although calpain and calpastatin genes behaved as housekeeping genes, changes in their expression mimicked apparent changes in proteolysis. The observation that calpain and calpastatin activities were unchanged indicates that additional regulation of the calpain:calpastatin system exists at other sites in muscle cells. To determine whether Dex-dependent changes in proteolysis were mediated indirectly, we assayed the effects of Dex on plasma thyroid hormone concentrations. Dex reduced both plasma triiodothyronine (T3) and thyroxine (T4) concentrations by as much as 68% and 67% respectively, and reduced the T3/T4 ratio by 74% following 4 days of Dex treatment. Thyroid hormones regulate muscle proteolysis and their lower concentrations in Dex-treated rabbits may account for the reductions in muscle NMH concentration and urinary NMH excretion caused by Dex.

Journal of Endocrinology (1994) 141, 209–217