bluuebunny/crossref_metadata_2025 huggingface dataset:
Sexual maturation in male rhesus monkeys: importance of neonatal testosterone exposure and social rank
Authors: Mann DR , Akinbami MA , Gould KG , Paul K , Wallen K
Published: December 2004

In a 5-year longitudinal study, we examined the effect of disrupting the neonatal activity of the pituitary-testicular axis on the sexual development of male rhesus monkeys. Animals in a social group under natural lighting conditions were treated with a GnRH antagonist (antide), antide and androgen, or both vehicles, from birth until 4 months of age. In antide-treated neonates, serum LH and testosterone were near or below the limits of detection throughout the neonatal period. Antide + androgen-treated neonates had subnormal serum LH, but above normal testosterone concentrations during the treatment period. From 6 to 36 months of age, serum LH and testosterone were near or below the limits of detection. Ten of 12 control animals reached puberty during the breeding season of their 4th year, compared with five of 10 antide- and three of eight antide + androgen-treated animals. Although matriline rank was balanced across treatment groups at birth, a disruption within the social group during year 2 resulted in a marginally lower social ranking of the two treated groups compared with the controls. More high (78%) than low (22%) ranking animals reached puberty during year 4. During the breeding season of that year, serum LH, testosterone and testicular volume were positively correlated with social rank. Thus the lower social rank of treated animals may have contributed to the subnormal numbers of these animals reaching puberty during year 4. However, of those animals achieving puberty during year 4, the pattern of peripubertal changes in serum testosterone and testicular volume differed between control and antide-treated animals. The results appear to suggest that the disruption of normal activity of the neonatal pituitary--testicular axis retarded sexual development, but that social rank is a key regulatory factor in setting the timing of sexual maturation in male rhesus monkeys. The effect of neonatal treatment with antide and low social rank on sexual development could not be reversed by neonatal exposure to greater than normal concentrations of androgen.
Antigoitrogenic effect of combined supplementation with dl-alpha-tocopherol, ascorbic acid and beta-carotene and of dl-alpha-tocopherol alone in the rat
Authors: Mutaku JF , Many MC , Colin I , Denef JF , van den Hove MF
Published: December 2004

The effects of the vitamins dl-alpha-tocopherol, ascorbic acid and beta-carotene, free radical scavengers and lipid peroxidation inhibitors, were analyzed in male Wistar rats made goitrous by feeding a low iodine diet (< 20 micrograms iodine/kg) and perchlorate (1% in drinking water) for 4, 8, 16, and 32 days. Groups of control or goitrous rats received for at least 16 days before killing a diet containing 0.6% vitamin E (as dl-alpha-tocopherol acetate), 1.2% vitamin C (ascorbic acid) and 0.48% beta-carotene, either simultaneously (vitamin cocktail) or separately. This treatment led to a 5-fold increase of vitamin E in the thyroid gland, a 24-fold increase in the liver and a 3-fold increase in the plasma. In control rats, vitamin cocktail administration increased slightly the thyroid weight with little changes in thyroid function parameters. During iodine deficiency, administration of the vitamin cocktail or vitamin E alone reduced significantly the rate of increase in thyroid weight, and DNA and protein contents, as well as the proportion of [3H]thymidine labeled thyroid follicular cells, but not that of labeled endothelial cells. Plasma tri-iodothyronine, thyroxine, TSH levels, thyroid iodine content and concentration as well as relative volumes of glandular compartments were not modified. The proportion of necrotic cells rose from 0.5% in normal animals to about 2% after 16 days of goiter development. No significant protective effect of the vitamins was observed. These results suggest that these vitamins, particularly vitamin E, modulate one of the regulatory cascades involved in the control of thyroid follicular cell growth, without interfering with the proliferation of endothelial cells.
Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail
Authors: Pawson AJ , Katz A , Sun YM , Lopes J , Illing N , Millar RP , Davidson JS
Published: December 2004

The chicken gonadotropin-releasing hormone receptor (GnRH-R) is notable for having a cytoplasmic C-terminal tail, which is not present in the mammalian GnRH-Rs. We report here that the cytoplasmic tail mediates rapid agonist-promoted receptor internalization. The chicken GnRH-R mediated internalization of gonadotropin-releasing hormone (GnRH) agonist (125I[His5-D-Tyr6]GnRH) at a rate of 11.3%.min-1, compared with only 0.71 %.min-1 for the human GnRH-R. To determine whether the presence of the cytoplasmic tail was responsible for the more rapid internalization kinetics of the chicken GnRH-R we truncated the tail after the Ile336 residue (S337stop). Receptor-mediated internalization of GnRH agonist by the S337stop-chicken GnRH-R was much slower than in the wild-type chicken receptor, and was similar to the wild-type human GnRH-R (0.55 %.min-1). These data indicate that rapid agonist-promoted internalization of the chicken GnRH-R is mediated through elements in the cytoplasmic C-terminal tail, distal to or including Ser337 and suggests that elimination of the C-terminal tail during evolution of mammalian GnRH-Rs may be related to its effects on internalization.
The effects of recombinant human IGF-I administration on concentrations of acid labile subunit, IGF binding protein-3, IGF-I, IGF-II and proteolysis of IGF binding protein-3 in adolescents with insulin-dependent diabetes mellitus
Authors: Cheetham TD , Holly JM , Baxter RC , Meadows K , Jones J , Taylor AM , Dunger DB
Published: December 2004

The long term therapeutic potential of recombinant human (rh) IGF-I administration in insulin-dependent diabetes mellitus (IDDM) may be determined by changes in the IGF binding proteins (IGFBPs) and thus the bioavailability of IGF-I. We have therefore studied the effects of a single subcutaneous dose of rhIGF-I (40 micrograms/kg at 1800 h), when compared with an untreated control night, in 17 subjects with IDDM, on serum concentrations of IGF-I, IGF-II, IGFBP-3, acid labile subunit (ALS), and IGFBP-3 proteolysis. Mean (+/- S.E.M.) IGF-I levels increased from 242 +/- 30 ng/ml to 399 +/- 26 ng/ml (P = 0.01) after rhIGF-I whereas IGF-II levels declined from 600 +/- 45 ng/ml to 533 +/- 30 ng/ml. There was a small overnight reduction in baseline ALS levels from 48 +/- 2.8 to 44.5 +/- 3.2 micrograms/ml (P = 0.04) after rhIGF-I administration. An early fall in IGFBP-3 concentrations on the control night was not seen after rhIGF-I and overall mean levels were increased (5.2 +/- 0.2 micrograms/ml vs 4.9 +/- 0.2 micrograms/ml, P = 0.04, on the control night). On the baseline night, IGFBP-3 levels correlated with the sum of IGF-I and IGF-II (r = 0.73, P = 0.02) and with levels of the ALS (r = 0.7, P = 0.002). However after rhIGF-I, the sum of IGF-I and IGF-II no longer correlated with IGFBP-3, whereas the relationship with ALS was maintained. Immunoblot studies in six subjects indicated that 60%-70% of the IGFBP-3 was detected as a low molecular weight fragment at 1900 h on both study nights, but the amount of fragment declined to approximately 50% at 0100 h and 45% at 0700 h. In conclusion, despite a slight but significant fall in ALS, IGFBP-3 levels rise after rhIGF-I administration in IDDM. This cannot be explained by alterations in IGFBP-3 proteolysis, and may relate to the relative stability of ALS/IGFBP-3 when complexed principally with IGF-I rather than IGF-II.
Stress responsiveness of the pituitary-interrenal axis during early life stages of common carp (Cyprinus carpio)
Authors: Stouthart AJ , Lucassen EC , van Strien FJ , Balm PH , Lock RA , Wendelaar Bonga SE
Published: December 2004

Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.
Insulin-like growth factor-binding protein-3 protease activity in Snell normal and Pit-1 deficient dwarf mice
Authors: Koedam JA , Hoogerbrugge CM , van Buul-Offers SC
Published: December 2004

Partial proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) lowers its affinity for IGFs. Presumably, this leads to destabilization of the ternary IGF-IGFBP-3-acid-labile subunit complex in the circulation and an increased bioavailability of IGFs. We investigated the effect of GH on IGFBP-3 proteolysis by comparing serum from normal mice and GH-deficient dwarf mice. While normal mouse serum degraded 125I-IGFBP-3, this activity declined with age. In contrast, serum from dwarf mice displayed strong proteolytic activity at all ages tested (up to 10 weeks). In dwarf mice of 4 weeks and older, this activity could not be inhibited by EDTA and 1,10-phenanthroline, indicating the presence of a divalent cation-independent protease. Prolonged treatment with GH (4 weeks) did not decrease the overall potency of the serum to degrade IGFBP-3, but partially restored the ability of EDTA to inhibit IGFBP-3 protease activity. GH deficiency therefore appears to induce a new kind of IGFBP-3 protease. Similarly, serum from hypophysectomized rats displayed enhanced IGFBP-3 protease activity compared with control rat serum. These results suggest that a protease induced under conditions of severe GH deficiency may contribute to making IGFs optimally available to the tissues.
Leydig cell apoptosis in the rat testes after administration of the cytotoxin ethane dimethanesulphonate: role of the Bcl-2 family members
Authors: Taylor MF , Woolveridge I , Metcalfe AD , Streuli CH , Hickman JA , Morris ID
Published: December 2004

Ethane dimethanesulphonate (EDS) is cytotoxic to Leydig cells in the adult rat. To investigate the role and regulation of apoptosis in the Leydig cell, EDS (100 mg/kg i.p.) was administered to adult male rats and the testes examined 6, 12, 18, 24, 48 and 72 h later. Numbers of Leydig cells, identified by 3 beta-hydroxysteroid dehydrogenase immuno-histochemistry started to fall by 12 h after EDS injection and were almost undetectable by 72 h. Apoptotic cells in the interstitium, visualised by in situ end labelling of DNA, increased in number to reach a maximum 24 h after injection of EDS, and were undetectable by 72 h. In many tissues the apoptosis-related gene products act in cohort: Bcl-2 and Bcl-xl promoting survival of a cell, whilst Bax promotes cell death often positively regulated by the tumour-suppressor gene p53. Western blot analysis showed that: (1) Bcl-2 and p53 were absent from interstitial Leydig cells but were expressed in the seminiferous tubules. (2) Bax protein although expressed in the interstitium was not present in the Leydig cells. (3) Bcl-xl in Leydig cells was transiently increased after EDS. In conclusion, EDS kills Leydig cells by apoptosis; however the control of Leydig cell death does not involve p53 or the Bcl-2 family members but may require other gene products yet to be identified.
Mutation and expression analysis of the cyclin-dependent kinase inhibitor gene p27/Kip1 in pituitary tumors
Authors: Takeuchi S , Koeffler HP , Hinton DR , Miyoshi I , Melmed S , Shimon I
Published: December 2004

By regulating cyclin-cyclin-dependent kinase (CDK) complex activity, individual CDK inhibitors (CDKIs) are potential tumor suppressors. One of the CDKIs, p27/Kip1, binds to a variety of CDK-cyclin complexes. A link between loss of p27/Kip1 function and development of pituitary tumors was suggested by the formation of pituitary tumors in almost all mice with germline deletion of the p27/Kip1 gene. However, genetic aberrations in the p27/Kip1 locus have not been analyzed in human pituitary tumors. We investigated eighteen non-functioning and GH-secreting pituitary tumor samples for p27/Kip1 mutations by single-strand conformational polymorphism (SSCP) following PCR. We found five abnormally migrating samples on the PCR-SSCP analysis. The sequence of these samples revealed a polymorphism of codon 109 (Val-->Gly), which has been previously described. No other structural changes of p27/Kip1 were found in these pituitary tumors within the coding region. In addition, no difference in p27/Kip1 protein levels in pituitary tumor tissues compared with normal pituitary tissues was demonstrated by immunostaining. These data suggest that both p27/Kip1 mutations and decreases in p27/Kip1 protein levels are infrequent in the development of pituitary tumors.
Hormonal regulation of connexin-43 in baboon corpora lutea
Authors: Khan-Dawood FS , Yang J , Dawood MY
Published: December 2004

The synthesis and secretion of progesterone in the corpus luteum are regulated by both endocrine and paracrine/ autocrine factors which affect the steroidogenic cells. Evidence suggests that these cells communicate via cell-cell junctional proteins, the connexins. Previously we have shown that connexin-43 is expressed in both human and baboon (Papio hamadryus anubis) corpora lutea, with differential expression throughout luteal development, but is not detectable in corpora albicantia. We have examined the effect of human chorionic gonadotropin (hCG), oxytocin, clomiphene citrate and the anti-progesterone onapristone on expression of connexin-43 protein in the early luteal phase 1-5 days after the mid-cycle luteinizing hormone (LH) surge (LH+ 1-5 days), the mid-luteal phase 6-10 days after the LH surge (LH+ 6-10 days), and the late luteal phase 11-15 days after the LH surge (LH+ 11-15 days) in corpora lutea obtained from normal adult cycling females. Connexin-43 was localized by immunohistochemistry in cultured cells from all the three stages. Western blot analysis of the treated cells indicated the presence of two bands at 43 and 45 kDa. The band at 45 kDa was found to be phosphorylated connexin-43, indicating the presence of functional gap junctions. hCG (10 IU/ml) stimulated the expression of connexin-43 throughout luteal development; however, maximum expression occurred in the early luteal phase with a significantly greater expression of the non-phosphorylated protein. In contrast, in the mid-luteal phase, the expression of the phosphorylated protein was predominant. Oxytocin (200 mU/ml) also stimulated connexin-43 expression throughout luteal development with similar effects on the phosphorylated and non-phosphorylated protein in the early and mid-luteal phase; however, compared with hCG, oxytocin had a greater effect on mid-luteal phase connexin-43 expression. In the presence of both hCG and oxytocin, the expression of connexin-43 was significantly higher than the control only in the late luteal phase. Both clomiphene citrate and onapristone suppressed connexin-43 expression, and concomitant addition of hCG did not counteract their effect. In the context of our previous studies, it is concluded that, together with LH/hCG and the steroid hormones, oxytocin is involved in cell-cell contact-dependent communication in the corpus luteum.
Effect of long-term glucocorticoid treatment on oestradiol-induced proliferation in the uterus of ovariectomized rats
Authors: Gunin AG
Published: December 2004

The aim of this study was to examine the effect of long-term treatment with glucocorticoids on the uterine response to oestradiol. Ovariectomized rats were treated with crystal triamcinolone acetonide (0.1 mg/100 g, i.m.) or saline (0.1 ml/100 g i.m.) for 29 days. Over this period five injections were administered, one per week. On the second day after the last triamcinolone injection, rats were treated with a single injection of oestradiol dipropionate (5 micrograms/100 g, s.c.) or vehicle (olive oil, 0.1 ml/100 g, s.c.). The effects of oestradiol in the uterus were determined by measuring mitotic index, bromodeoxyuridine (BrdU)-labelling index (BrdU was injected 2 h before the rats were killed; 2 mg/100 g, i.p.), and proliferating cell nuclear antigen (PCNA)-labelling index 24, 36 and 48 h after the injection of oestradiol or vehicle. Long-term treatment with glucocorticoids resulted in dissimilar changes in oestradiol-induced proliferation in epithelial and connective-tissue (stroma) components of the uterus. In luminal and glandular epithelia, there was an initial reduction in proliferation at 24 h, followed by an increase at 36 h and a further reduction at 48 h after the oestradiol injection. In stromal cells of the endometrium, triamcinolone treatment caused a large constant increase in oestradiol-induced proliferation throughout the experiment. The glucocorticoid treatment had no effect on the parameters without oestradiol administration.
Cardiac protein kinase C expression in two models of cardiac hypertrophy associated with an activated cardiac renin-angiotensin system: effects of experimental hyperthyroidism and genetic hypertension (the mRen-2 rat)
Authors: Fryer LG , Holness MJ , Decock JB , Sugden MC
Published: December 2004

There is evidence for a role of protein kinase C (PKC) in the development of cardiac hypertrophy. We examined the expression of individual PKC isoforms in the adult rat heart in two distinct, well-characterised in vivo models of cardiac hypertrophy associated with an activated cardiac renin-angiotensin system, namely experimental hyperthyroidism and the TGR(mRen2)27 rat. The cardiac expression of a range of PKC isoforms (PKC-alpha, PKC-omega, PKC-epsilon, PKC-gamma, and PKC-tau) was examined by immuno-blotting. Our work demonstrates that the expression of total cardiac nPKC-omega and nPKC-epsilon relative to protein is selectively and differentially modified in these models. A consistent up-regulation of nPKC-omega in conjunction with overall down-regulation of nPKC-epsilon was observed in both models. The expression of other PKC isoforms was unaffected. The divergent responses of the expression of the two nPKC isoforms to an activated cardiac renin-angiotensin system in vivo in adulthood suggest that these individual nPKC isoforms subserve specific roles in the response.
Insulin-like growth factor I reduces thyroid hormone receptors in the rat liver. Evidence for a feed-back loop regulating the peripheral thyroid hormone action
Authors: Pellizas CG , Coleoni AH , Costamagna ME , Di Fulvio M , Masini-Repiso AM
Published: December 2004

Tri-iodothyronine (T3) is known to be involved in the regulation of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis. In previous studies we demonstrated that IGF-I and GH reduced the metabolic response to T3 measured as the activity of two T3-dependent enzymes, mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) in cultured rat liver cells. In this study we analysed in vivo the effect of IGF-I administered to rats on the activity of alpha-GPD and ME. IGF-I (240 micrograms/100 g body weight (BW) every 12 h for 48 h) significantly diminished alpha-GPD (P < 0.01) and ME (P < 0.05) activities. Serum basal glucose concentration was not significantly modified 12 h after the administration of recombinant human IGF-I (240 and 480 micrograms/100 g BW every 12 h for 48 h). Under similar conditions, no significant change in serum total thyroxine (TT4) concentration was observed, although free thyroxine (FT4) was diminished (P < 0.02) and total T3 (TT3) was increased (P < 0.03). To explore the participation of the nuclear thyroid hormone receptor (THR) in the mechanism of IGF-I action we measured the maximal binding capacity and the affinity constant (Ka) of THR by Scatchard analysis, and concentrations of messenger RNAs (mRNAs) that code for the isoforms of THR present in the liver (beta 1, alpha 1 and alpha 2) by Northern blot. IGF-I (240 micrograms/100 g BW every 12 h for 48 h) significantly reduced maximal binding capacity to 37% of the control value (P < 0.01) without changes in the Ka. beta 1, alpha 1 and alpha 2 THR mRNAs were significantly reduced (P < 0.01) by 120-480 micrograms/100 g BW IGF-I administration every 12 h for 48 h. Time-course studies indicated that this effect was obtained 12 h after the administration of 240 micrograms/100 g BW IGF-I (P < 0.05). These results indicate that IGF-I administration to rats diminishes the metabolic thyroid hormone action in the liver by a mechanism that involves, at least in part, a reduction in the number of THRs and in their level of expression.
Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts
Authors: Kraaij R , Verhoef-Post M , Grootegoed JA , Themmen AP
Published: December 2004

Glycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormone (FSH) receptor gene: FSH-R1 and FSH-R2. The splice variant FSH-R1 differs from the full-length FSH receptor mRNA by the inclusion of a small extra exon between exons 9 and 10. FSH-R2 lacks the first three base pairs of exon 4, contains an extra exon between exons 4 and 5, and has an extended 3'-untranslated region. According to the predicted open reading frames, both mRNAs encode truncated FSH receptor proteins, consisting of the entire extracellular domain (FSH-R1) or the amino-terminal half of the extracellular domain (FSH-R2), and are expressed at a low level in testes and ovaries. The levels of expression of the FSH-R1 and FSH-R2 mRNAs in the gonads show a constant ratio to the expression level of the full-length FSH receptor mRNA. Furthermore, in vitro co-expression of either one of the truncated proteins with the full-length FSH receptor in COS1 cells did not affect signal transduction through the full-length FSH receptor. The absence of a function of the truncated FSH receptors in FSH signal transduction in vitro, and the lack of differential regulation of the alternative transcripts, indicate that there is no clear function for alternative splicing of the FSH receptor pre-mRNA in the postnatal testis and the cycling adult ovary.
Expression of prolactin mRNA and of prolactin-like proteins in endothelial cells: evidence for autocrine effects
Authors: Clapp C , Lopez-Gomez FJ , Nava G , Corbacho A , Torner L , Macotela Y , Duenas Z , Ochoa A , Noris G , Acosta E , Garay E , Martinez de la Escalera G
Published: December 2004

Formation of new capillary blood vessels, termed angiogenesis, is essential for the growth and development of tissues and underlies a variety of diseases including tumor growth. Members of the prolactin hormonal family bind to endothelial cell receptors and have direct effects on cell proliferation, migration and tube formation. Because many angiogenic and antiangiogenic factors are produced by endothelial cells, we investigated whether endothelial cells expressed the prolactin gene. Here we show that bovine brain capillary endothelial cells (BBCEC) in culture express the full-length prolactin messenger RNA, in addition to a novel prolactin transcript, lacking the third exon of the gene. In addition cultures of BBCEC synthesize and secrete prolactin-like immunoreactive proteins with apparent molecular masses of 23, 21 and 14 kDa. The prolactin-like nature of these proteins in supported by the observation that Nb2-cells, a prolactin-responsive cell line, were stimulated to proliferate when co-cultured with endothelial cells and this stimulation was neutralized with prolactin-directed antibodies. Finally, consistent with a possible autocrine effect of endothelial-derived prolactins, polyclonal and monoclonal prolactin antibodies specifically inhibited basal and basis fibroblast growth-factor-stimulated growth of endothelial cells. Taken together, the present findings support the hypothesis of the prolactin gene being expressed in endothelial cells as proteins that could act in an autocrine fashion to regulate cell proliferation.
Studies on the role of ACTH in the regulation of adrenal responsiveness and the timing of parturition in the ovine fetus
Authors: Poore KR , Young IR , Canny BJ , Thorburn GD
Published: December 2004

A dramatic late-gestation increase in fetal plasma cortisol concentrations is critical for the timing of parturition in the sheep. This increase appears to depend upon an intact hypothalamo-pituitary unit and is characterised by increasing responsiveness of the fetal adrenal gland to ACTH. ACTH has been postulated as the critical determinant of the late-gestation cortisol increase; however, recent evidence has suggested that other factors, including the ACTH precursor, pro-opiomelanocortin, may also be involved. To further define the role of ACTH in determining the timing of parturition and the responsiveness of the fetal adrenal gland, intact (INT/ACTH) and hypophysectomised (HX/ACTH) fetuses received a continuous infusion of ACTH(1-24) from the time of surgery (approximately 115 days gestational age (GA)) at a rate we have previously shown to generate normal fetal cortisol concentrations and term parturition in HX fetuses. A third group of saline-infused intact fetuses (INT/SAL) served as the control group. Adrenal responsiveness was assessed by cortisol responses to ACTH(1-24) challenges at 120, 130 and 140 days GA. There were no differences between the three groups of fetuses in the timing of parturition, the late-gestation increase in cortisol concentrations or the size of the adrenal cortex. In both INT/SAL and INT/ACTH fetuses, there were significant increases in basal immunoreactive-ACTH concentrations with advancing GA, although no such increase was observed in HX/ACTH fetuses. The proportion of total ACTH immunoreactivity present in low molecular weight (LMW) forms in INT/ACTH fetuses was greater than that in INT/SAL fetuses, while the level of LMW ACTH in HX/ACTH fetuses was intermediate. Both ACTH(1-24)-infused groups of fetuses had dramatically enhanced adrenal responsiveness to ACTH(1-24) at all GAs tested when compared with INT/SAL fetuses and there was a correlation (in rank order) between the proportion of LMW ACTH immunoreactivity and adrenal responsiveness. From these observations it appears that there is a separate regulation of adrenal responsiveness from basal cortisol concentrations and that an increase in basal cortisol concentrations can occur in the absence of an increase in basal ACTH concentrations. Furthermore, an increase in adrenal responsiveness does not appear to predict the timing of parturition nor basal cortisol concentrations. Taken together with previous studies it appears that ACTH plays an essential role in maintaining the growth of the fetal adrenal and enhancing its responsiveness, but a late-gestation increase in ACTH concentrations is not required to regulate basal cortisol concentrations or the timing of parturition.
Regulation of oxytocin receptor in the placentome capsule throughout pregnancy in the ewe: the possible role of oestradiol receptor, progesterone receptor and aromatase
Authors: Leung ST , Reynolds TS , Wathes DC
Published: December 2004

The hormonal regulation of uterine oxytocin receptors (OTR) during the establishment of pregnancy and at parturition has been studied extensively, but little information is available during mid-pregnancy. This study investigated the localisation of OTR mRNA in the ovine placentome throughout gestation and related this to expression patterns for the putative regulatory agents aromatase, oestradiol receptor, progesterone receptor and oxytocin. Placentomes were collected at regular intervals throughout pregnancy for in situ hybridisation analysis and immunocytochemistry (oestradiol receptor only). Results were quantified by optical density measurements of autoradiographs. Progesterone receptor mRNA was localised to the caruncular tissues on day 30 but became undetectable by day 34. Aromatase mRNA appeared in the fetal villi at days 34-40, with concentrations peaking at days 52-55 and again at days 132-137. Oestradiol receptor mRNA was localised to the caruncular tissues from days 13 to 30 and found in the maternal villi and placentome capsule from days 45 to 70. Oestradiol receptor protein was barely detectable in either tissue. OTR mRNA was localised to the placentome capsule at days 34-40, remaining high at day 45 and declining to basal levels by days 132-137. Oxytocin mRNA was not detected in the placentome. In conclusion: (1) progesterone acting via its receptor may suppress the expression of aromatase and OTR in early pregnancy; (2) the up-regulation of OTR expression in the capsule may not involve the oestradiol receptor; (3) there is a differential regulation between different regions of the uterus as the increase in the placentome capsule occurs at a time when concentrations in the rest of the endometrium and myometrium remain low; (4) oestradiol receptor expression in the placentome may be regulated at the translational level; and (5) there is no local production of oxytocin in the sheep placenta. The role of ORTs in the capsule during mid-pregnancy remains to be determined.
Effect of nitric oxide on rat adrenal zona fasciculata steroidogenesis
Authors: Cymeryng CB , Dada LA , Podesta EJ
Published: December 2004

The present study was designed to investigate the role of nitric oxide (NO) in the regulation of adrenocortical function. Different NO donors, such as sodium nitroprusside (SNP), S-nitroso-L-acetyl penicillamine, diethylamine/NO complex sodium salt and diethylenetriamine NO adduct, significantly decreased corticosterone production both in unstimulated and in corticotropin-stimulated zone fasciculata adrenal cells, in a dose-dependent manner. The effect of SNP was reversed by ferrous hemoglobin. A selective inhibitor of NO synthase, L-NG-nitro-arginine significantly increased corticosterone secretion. The effect of SNP was not mediated by cGMP as permeable cGMP analogs did not reproduce its inhibitory effect. SNP significantly inhibited the steroidogenesis stimulated by 8Br-cAMP and 22(R)-OH-cholesterol, but was ineffective when corticosterone was produced in the presence of exogenously added pregnenolone. Moreover, the conversion of [3H]cholesterol to [3H]pregnenolone and the production of pregnenolone or progesterone (assessed by RIA) were significantly decreased by SNP. Taken together, these results suggest that NO may be a negative modulator of adrenal zona fasciculata steroidogenesis.
Oogenesis in Atlantic salmon (Salmo salar L.) occurs by zonagenesis preceding vitellogenesis in vivo and in vitro
Authors: Celius T , Walther BT
Published: December 2004

Fish oogenesis represents pleiotropic cytodifferentiative programs including hepatic synthesis of the molecular components for both the eggshell and the oocytic energy deposits. Both hepatic processes are directly controlled by plasma levels of estradiol (E2), and injected E2 induces both biogenetic processes in prepubertal fish of both sexes. This work compares the temporal pattern of E2-induced biosynthesis of zona radiata proteins (zr-proteins) and vitellogenin in Atlantic salmon (Salmo salar L.) in vivo and in vitro. We monitored the presence of plasma zr-proteins and vitellogenin, using homologous polyclonal antiserum to zr-proteins and a monoclonal antibody to vitellogenin. Zr-proteins were induced by all E2 concentrations (0.001-1.1 mg/kg body weight (bw)) within one week of exposure while vitellogenin was not induced until two weeks post-injection and then only in plasma from fish injected with high E2 concentrations (0.4 mg or 1.1 mg/kg bw). After E2 treatment, hepatocytes isolated from male fish synthesized zr-proteins and vitellogenin in vitro. However, zr-proteins were secreted into the medium two days before vitellogenin, as measured by ELISA. The data indicate a preferential induction of zr-proteins compared with vitellogenin, both with regard to E2 sensitivity and response time to E2 treatment. These findings suggest an obligate sequence in salmon oogenesis. During sexual maturation low E2 levels at first induce only zonagenesis, while increasing levels of E2 subsequently induce both zonagenesis and vitellogenesis. In nature, the interval between zonagenesis and vitellogenesis may, therefore, be considerable. The data suggest new control mechanisms in fish oogenesis.
Regulation of the GTP-binding protein-based antilipolytic system of sheep adipocytes by growth hormone
Authors: Doris RA , Kilgour E , Houslay MD , Vernon RG
Published: December 2004

Chronic exposure of sheep adipose tissue to growth hormone (GH) in vitro decreases the ability of the adenosine analogue, N6-phenylisopropyladenosine (PIA), to inhibit isoprenaline-stimulated lipolysis by a mechanism which is dependent on both gene transcription and protein serine/threonine phosphorylation. The inhibition is not due to a change in ligand binding to the adenosine receptor, the amounts of the three isoforms of the inhibitory GTP-binding protein, Gi, or the maximum (forskolin-stimulated) adenylate cyclase activity. The ability of GH to modulate the PIA-activated adenosine receptor to stimulate dissociation of heterotrimeric Gi was assessed by measurement of pertussis toxin-catalysed ADP-ribosylation of Gi; GH does not appear to alter the interaction between the activated receptor and Gi. The ability of GH to alter the ability of activated Gi to inhibit adenylate cyclase activity was assessed by measuring the ability of a GTP analogue, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), to inhibit forskolin-stimulated adenylate cyclase activity; chronic exposure to GH prevented this effect of p[NH]ppG. Thus the attenuation of the inhibition of lipolysis by PIA by chronic exposure of adipocytes to GH appears to be due to an impairment in the interaction between adenylate cyclase and the alpha subunit of one or more isoforms of Gi.
Modulation of human thyrotropin oligosaccharide structures--enhanced proportion of sialylated and terminally galactosylated serum thyrotropin isoforms in subclinical and overt primary hypothyroidism
Authors: Trojan J , Theodoropoulou M , Usadel KH , Stalla GK , Schaaf L
Published: December 2004

Enhanced sialylation of thyrotropin (TSH) prolongs its metabolic clearance rate and thus increases the hormone's in vivo bioactivity. This has been shown for hypothyroid rats and for recombinant human TSH, but there are few data on the sialylation of human serum TSH. The aim of this work was to further study sialylated human serum TSH, its precursors bearing terminal galactose residues, and the role of pharmacological doses of thyrotropin-releasing hormone (TRH) on their secretion under different degrees of primary hypothyroidism. We analyzed serum TSH in patients with subclinical (n = 9) and overt primary hypothyroidism (n = 13) compared with euthyroid individuals (n = 12) and human standard pituitary TSH (IRP 80/558). Blood was drawn before and 30 min after intravenous administration of 200 micrograms TRH, and TSH was purified by immunoaffinity concentration. The content of sialylated (sialo-) TSH and isoforms bearing terminal galactose (Gal-TSH, asialo-Gal-TSH) was measured by Ricinus communis (RCA 120) affinity chromatography in combination with enzymatic cleavage of sialic acid residues. TSH immunoreactivity was measured by an automated second generation TSH immunoassay. Pituitary TSH contained 16.5 +/- 0.8% Gal-TSH. In euthyroid individuals the proportion of Gal-TSH was 14.6 +/- 1.9%, whereas TSH in patients with subclinical and overt primary hypothyroidism contained 23.9 +/- 3.5% (P < 0.05 vs euthyroid individuals) and 21.1 +/- 1.7% Gal-TSH respectively. The mean ratio of asialo-Gal TSH was 23.8 +/- 0.6% for pituitary TSH, 35.7 +/- 4.2% in euthyroid individuals, 48.0 +/- 3.3% in patients with subclinical, and 61.5 +/- 3.8% (P < 0.001 vs euthyroid individuals) in patients with overt primary hypothyroidism. For pituitary TSH the calculated proportion of sialo-TSH was 6.5 +/- 0.2%, for euthyroid individuals 20.3 +/- 2.8%, for patients with subclinical hypothyroidism 24.1 +/- 3.0%, and for patients with overt primary hypothyroidism 40.7 +/- 3.0% (P < 0.001 vs euthyroid individuals). The proportions of Gal-TSH, asialo-Gal-TSH, and sialo-TSH did not differ significantly before and after TRH administration in the individuals studied. Our data show that patients with subclinical and overt primary hypothyroidism have a markedly increased proportion of serum TSH isoforms bearing terminal galactose and sialic acid residues, which may represent a mechanism for the further stimulation of thyroid function. Pharmacological doses of TRH cause an increased quantity of TSH to be released, but do not significantly alter the proportion of sialylated or terminally galactosylated TSH isoforms.
Type I collagen influence on gene expression in UMR106-06 osteoblast-like cells is inhibited by genistein
Authors: Celic S , Katayama Y , Chilco PJ , Martin TJ , Findlay DM
Published: December 2004

We have previously shown that an exogenous type I collagen matrix can regulate expression of mRNA for parathyroid hormone (PTH)-related protein (PTHrP) and its receptor, the PTH/PTHrP receptor, in the UMR106-06 osteogenic sarcoma cell line, which is considered to be representative of a relatively mature osteoblast phenotype. Consistent with those data, we show here that growth of UMR106-06 cells on type I collagen increased PTH/PTHrP receptor-binding capacity. Analysis of the binding data showed that the number of PTH/PTHrP receptors expressed by cells cultured on collagen was at least 2-fold greater than that of cells cultured on plastic. Expression of mRNA encoding alkaline phosphatase (ALP) and osteopontin (OP) was also upregulated in cells cultured on collagen, suggesting that interaction with collagen promotes the osteoblast phenotype in this cell line. Retinoic acid (RA), which has also been shown to promote osteoblastic differentiation, synergized with type I collagen to cause super-induction of OP mRNA. In contrast, RA abolished the collagen-induced increase in ALP mRNA and PTH/PTHrP receptor mRNA. The collagen-mediated increase in the expression of OP and PTH/PTHrP receptor mRNA, but not that of ALP, was perturbed by prior covalent modification of the collagen by non-enzymatic glycation. The collagen effects did not occur via interaction with RGD amino acid domains in type I collagen, but evidence was obtained for involvement of the DGEA amino acid cell-binding domain. The mechanism by which plating of UMR106-06 cells on a type I collagen substrate affects PTH/PTHrP receptor mRNA levels was investigated. Inhibition of cytoskeletal organization using cytochalasin D, and inhibitors of protein phosphatases, protein kinase C, phospholipase C and cyclooxygenase, did not abrogate the collagen-mediated effects. In contrast, treatment of cells with the protein tyrosine kinase inhibitor genistein, but not herbimycin A, dose-dependently abolished the collagen effects on the expression of PTH/PTHrP receptor, ALP and OP mRNA. These results show that a type I collagen substrate influences the expression of osteoblast-associated genes in a cell model of mature osteoblasts and suggests that this involves, at least in part, changes in intracellular tyrosine phosphorylation.
Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo
Authors: Hakola K , Haavisto AM , Pierroz DD , Aebi A , Rannikko A , Kirjavainen T , Aubert ML , Huhtaniemi I
Published: December 2004

We have previously described the preparation, purification and partial characterization of recombinant (rec) forms of rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the present study, the special functional features of these hormones were studied further, in vitro and in vivo, and compared with human recLH and recFSH, as well as with human urinary choriongonadotropin (hCG) and rat pituitary LH (NIDDK-RP3). In radioreceptor assay, the affinity of hCG binding to rat testis membranes was 5-fold higher than that of human recLH and 100-fold higher than that of rat recLH. In in vitro bioassay, using dispersed adult mouse interstitial cells or a mouse Leydig tumor cell line (BLT-1), hCG and human recLH were 10- to 20-fold more potent than rat recLH. Correspondingly, rat pituitary LH was about 10-fold less potent than rat recLH, and evoked a maximum testosterone response that was about half of that elicited by the other LH/CG preparations. Rat recFSH was about 10-fold less potent than human recFSH in stimulating cAMP production of a mouse Sertoli cell line (MSC-1) expressing the recombinant rat FSH receptor. The circulating half-times (T1/2) of rat and human rec hormones were assessed after i.v. injections into adult male rats rendered gonadotropin-deficient by treatment with a gonadotropin-releasing hormone antagonist. A novel immunometric assay was used for the rat FSH measurements. In the one-component model the T1/2 values of rat and human recLH were 18.2 +/- 1.9 min (n = 7) and 44.6 +/- 3.1 min (n = 7) respectively and those of rat and human recFSH were 88.4 +/- 10.7 min (n = 6) and 55.0 +/- 4.2 min (n = 6) respectively; the two-component models revealed similar differences between the rec hormone preparations. Collectively, rat recLH was eliminated significantly faster from the circulation than human recLH (P < 0.0001). In contrast, the elimination of rat recFSH was significantly slower than that of human recFSH (P = 0.02). In conclusion, rat recFSH and rat recLH display lower biopotencies per unit mass than the respective human hormones in vitro, and also in vivo for LH. This is paralleled by shorter T1/2 of rat recLH than the respective human hormone in the circulation, whereas human recFSH has a shorter T1/2 than human FSH. The special functional features of the rat rec gonadotropins emphasize the use of these preparations on studies of gonadotropin function in the rat, an important animal model for reproductive physiology.
Effects of colony stimulating factor-1 on human extravillous trophoblast growth and invasion
Authors: Hamilton GS , Lysiak JJ , Watson AJ , Lala PK
Published: December 2004

Colony stimulating factor (CSF)-1 has been localized in a variety of tissues and shown to influence proliferation and differentiation of numerous cell types. Messenger RNA and protein products of CSF-1 and its receptor (c-fms) have been identified in the human placenta and decidua. We examined whether CSF-1 and c-fms mRNA and protein are expressed by normal human first trimester invasive extravillous trophoblast (EVT) cells propagated in culture and whether CSF-1 influences proliferation and/or invasion of these cells. CSF-1 mRNA and protein expression was determined by RT-PCR and immunofluorescence microscopy. Proliferation was assessed by the cellular uptake of tritiated thymidine and invasion was evaluated by Matrigel invasion assay as well as Northern blot analysis of mRNA expression for invasion-associated enzymes and their inhibitors. Results revealed that normal invasive EVT cells in culture express both CSF-1 and c-fms mRNA and protein. Under serum-free conditions, exogenous CSF-1 greatly stimulated the proliferation of these cells. CSF-1 neutralizing and c-fms receptor blocking antibody (Ab) each abolished the growth stimulatory effects of CSF-1, indicating that CSF-1 and c-fms interaction was responsible for these effects. In fact, c-fms Ab alone reduced proliferation to below background levels. While exogenous CSF-1 failed to influence EVT cell invasiveness, Northern blot analysis of mRNA indicated a slight upregulation of the invasion-associated enzyme 72 kDa type IV collagenase as well as its natural inhibitor tissue inhibitor of metalloprotease (TIMP)-1, so that the balance between the two remained unaltered. These findings suggest that CSF-1 may represent an autocrine (and possibly paracrine) growth stimulatory factor for the invasive trophoblast cells in situ with no net effect on their invasiveness.
Leptin alters the response of the growth hormone releasing factor- growth hormone--insulin-like growth factor-I axis to fasting
Authors: LaPaglia N , Steiner J , Kirsteins L , Emanuele M , Emanuele N
Published: December 2004

Proper nutritional status is critical for maintaining growth and metabolic function, playing an intimate role in neuroendocrine regulation. Leptin, the recently identified product of the obese gene, may very well be an integral signal which regulates neuroendocrine responses in times of food deprivation. The present study examines leptin's ability to regulate hormonal synthesis and secretion within the GRF-GH-IGF axis in the adult male rat during almost 3 days of fasting. Serum levels of GH and IGF-I were drastically suppressed by fasting. Daily leptin administration was able to fully prevent the fasting-induced fall in serum GH. Leptin failed to restore IGF-I to control levels, however, suggesting possible GH resistance. Fasting caused an insignificant increase in GH mRNA, while leptin injections significantly increased steady-state levels of this message. The GRF receptor (GRFr) message was not altered with fasting or leptin treatment. Leptin also exhibited effects at the hypothalamic level. Fasting induced a sharp fall in GRF mRNA expression and leptin injections partially prevented this fall. However, there were no observed changes in the hypothalamic GRF content. These results provide evidence that leptin may function as a neuromodulator of the GRF-GH-IGF axis communicating to this hormonal system the nutritional status of the animal.