bluuebunny/crossref_metadata_2025 huggingface dataset:
Autocrine inhibition of milk secretion in the lactating tammar wallaby (Macropus eugenii)
Authors: Hendry KA , Simpson KJ , Nicholas KR , Wilde CJ
Published: December 2004

The lactating tammar wallaby progressively alters the rate of secretion and composition of its milk to provide appropriate nutrition for the developing offspring, whose needs are signalled by changes in the pattern and efficiency of its sucking. Tammars are also capable of asynchronous concurrent lactation, when the mother provides a dilute milk for a newborn young permanently attached to the teat (phase 2A of lactation), and a concentrated milk from an adjacent mammary gland for a young-at-heel (phase 3). The relationship between suckling behaviour and milk secretion, and the ability of adjacent glands to function independently, suggests that milk secretion is controlled locally, within each mammary gland, by a mechanism sensitive to frequency and completeness of milk removal. To determine if tammar milk contains a factor able to control milk secretion, milk fractions have been screened in tissue and cell culture bioassays. A 6-30 kDa fraction of phase 3 whey was found to inhibit milk constituent synthesis and secretion in vitro, and inhibitory activity was associated with two discrete fractions obtained by anion exchange chromatography, which contained protein bands migrating anomalously at 66 kDa and 63 kDa in SDS-PAGE. These bands were recognised in Western blotting by antiserum raised against a bovine autocrine inhibitor of milk secretion. By the same criteria, milk secreted in phase 2B of tammar lactation, when milk secretion is low and suckling intermittent but less vigorous than phase 3, also contained a feedback inhibitor of milk secretion. The results indicate that, as in dairy animals, marsupial milk secretion is under local control through feedback inhibition by a milk protein, and raise the possibility that autocrine feedback may influence the transition from phases of low milk secretion (phase 2A, 2B) to a high rate in the final third phase of lactation.
Levels of corticotrophin-releasing hormone receptor subtype 1 mRNA in pregnancy and during labour in human myometrium measured by quantitative competitive PCR
Authors: Rodriguez-Linares B , Phaneuf S , Lopez Bernal A , Linton EA
Published: December 2004

It is suggested that corticotrophin-releasing hormone (CRH) is involved in parturition. We have previously reported the presence of the CRH receptor subtype 1 (CRH R1) in human uterine myocytes. The aim of the present study was to investigate whether expression of the CRH R1 in myometrial tissue changes in pregnancy and labour. We used a quantitative competitive PCR method to measure the mRNA levels of this receptor in non-pregnant and in term pregnant myometrium before and at different stages of labour. The levels of mRNA for the housekeeping gene for glucocerebrosidase (GCB) were also determined. The results were expressed as a ratio of CRH R1 and GCB mRNA levels. We have found that in pregnancy the CRH R1 is down-regulated from a ratio of 0.093+/-0.011 in non-pregnant myometrium to 0.012+/-0.005 (P<0.001) in term non-labouring myometrium. No significant changes were observed in the CRH R1:GCB ratio in tissues sampled within 13 h (0.013+/-0.004) from the start of labour. In summary, normalised levels of CRH R1 are down-regulated in pregnancy and do not change during labour. We speculate that our results do not support a direct role for the CRH R1 receptor in myometrial stimulation.
Effects of sex steroids on gonadotropin (FSH and LH) regulation in coho salmon (Oncorhynchus kisutch)
Authors: Dickey JT , Swanson P
Published: December 2004

The effect of steroid hormone treatment on coho salmon (Oncorhynchus kisutch) was examined. The cDNAs for coho salmon FSH beta and LH beta subunits were cloned and sequenced using reverse transcriptase PCR. Northern blot analysis revealed that a single transcript of 1 kb for each of these subunits was present in the pituitaries of vitellogenic and spermiating coho salmon. RNase protection assays (RPAs) were developed to quantify FSH beta and LH beta subunit transcript levels. For the RPAs, antisense RNA probes and sense RNA standards were prepared from a region of the cDNAs which spanned the signal peptide and a portion of the mature protein. These RPAs were used to examine the effects of exogenous steroids including testosterone, estradiol-17beta (E2) and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P) in vivo, in coho salmon at three time points during the spring period of gonadal growth when plasma levels of FSH are increasing. Both testosterone and E2 increased steady state mRNA levels of LH beta, whereas E2 decreased steady state mRNA levels of FSH beta in one experiment. Thus, the RPAs were able to detect changes in steady state mRNA levels in response to exogenous steroid treatment. Plasma and pituitary levels of FSH and LH were also measured using RIA. Throughout the experimental series, FSH plasma levels decreased in response to exogenous testosterone and E2 administration, while 17alpha,20beta-P had no effect on FSH plasma levels. Plasma LH levels were not detected throughout the course of the experiment. Pituitary LH increased in response to testosterone and E2, while pituitary FSH levels did not change. 17alpha,20beta-P had no effect on pituitary FSH or LH content during the experiment. Thus, regulation of the gonadotropins in coho salmon occurs at both the transcriptional as well as the translational level. Testosterone and E2 appear to have negative feedback effects on FSH, but positive feedback on LH.
Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris
Authors: Fidler AE , Lun S , Young W , McNatty KP
Published: December 2004

The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MFalpha) leader and ovine follicle stimulating hormone (oFSH) alpha and beta subunit coding sequences. Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51.0 ng/ml and 17.5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the alpha and one copy of the beta subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay). It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive. Measurements of secreted roFSH alpha and beta subunit concentrations indicated less than 10% of alpha and 25-33% of beta subunits were stably dimerized. The receptor binding properties of the roFSH resemble those of native oFSH. In summary this paper reports the production, by P. pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.
Pax-8 protein levels regulate thyroglobulin gene expression
Authors: Fabbro D , Pellizzari L , Mercuri F , Tell G , Damante G
Published: December 2004

Pax proteins are transcription factors that control differentiation of several cell types. In adult organisms Pax-8 is expressed in the follicular thyroid cell where it interacts with sequences of thyroglobulin and thyroperoxidase promoters. In this study, we provide evidence indicating that Pax-8 protein levels regulate thyroglobulin gene transcription. The most critical approach consisted in increasing Pax-8 protein levels by transfecting thyroid cells with a Pax-8 expression vector. In this situation the thyroglobulin promoter transcriptional activity was significantly increased with respect to untransfected cells. In contrast, the transfection of thyroid transcription factor-1 (TTF-1) expression vector causes a modest decrease of thyroglobulin promoter activity, rather than an increase. Northern blots of human papillary cancers reveal a significant correlation between Pax-8 and thyroglobulin mRNAs. Gel-retardation assays suggest that the mechanism by which the Pax-8 protein levels modulate thyroglobulin promoter activity may occur through competition with TTF-1 for a common binding site. Since we also demonstrate that Pax-8 expression is subjected to TSH control, our data strongly suggest that Pax-8 protein levels could represent an important determinant for the regulation of thyroid cells.
Cloning and characterisation of the gene encoding mole rat (Spalax ehrenbergi) growth hormone
Authors: Lioupis A , Nevo E , Wallis M
Published: December 2004

In mammals the structure of pituitary GH is generally strongly conserved, reflecting a slow basal rate of molecular evolution. However, on a few occasions the rate has increased - markedly during the evolution of primates and artiodactyls, and to a small extent during the evolution of rodents and rabbit - giving rise to marked differences between GH sequences of these species. In order to extend knowledge of rodent GHs we have cloned and characterised part of the GH gene of the Eurasian mole rat (Spalax ehrenbergi) using genomic DNA and a PCR technique. The sequence of all of the coding region and 5' untranslated region (UTR), most of the 3' UTR and part of the promoter region is described. The overall organisation of the mole rat GH gene is similar to that of GH genes from other mammals. The proximal Pit-1 sequence in the gene promoter differs somewhat from that of rat or mouse. The deduced sequence for the mature GH from mole rat differs from that of pig GH (thought to be identical to the ancestral placental mammal GH sequence) at 7 residues and from rat, mouse and hamster GHs at 9 to 12 residues. Only one or two of these substitutions involve residues close to the receptor-binding sites of the hormone.
Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus
Authors: Hiroi H , Inoue S , Watanabe T , Goto W , Orimo A , Momoeda M , Tsutsumi O , Taketani Y , Muramatsu M
Published: December 2004

In order to investigate the localization of estrogen receptor (ER) alpha and ERbeta in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ERalpha antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ERbeta antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ERalpha antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ERbeta antibody. In rat ovary, positive signals were shown with anti-ERbeta antibody in the nuclei of granulosacells. No specific immunostaining was observed with anti-ERalpha antibody. Although ERbeta was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ERbeta was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ERalpha and ERbeta in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs.
Insulin-glucocorticoid interactions in the regulation of acetyl-CoA carboxylase-alpha transcript diversity in ovine adipose tissue
Authors: Travers MT , Barber MC
Published: December 2004

Transcription of the acetyl-CoA carboxylase (ACC)-alpha gene is initiated from two promoters, promoter I (PI) and promoter II (PII) such that transcripts demonstrate heterogeneity in their 5' untranslated regions (UTR). Exons 1 and 2 (E1 and E2) are the primary exons in transcripts initiated from PI and PII respectively; E5 is the first coding exon present in all transcripts. In addition alternative exon splicing results in transcripts that either include or exclude a 47 nucleotide sequence corresponding to E4, such that E[1/4/5] and E[1/5] type transcripts result from PI activity, whereas transcripts containing E[2/4/5] or E[2/5] in the 5'UTR result from PII. In subcutaneous adipose tissue from non-pregnant non-lactating sheep approximately 60% of ACC-alpha transcripts are derived from PI, of which 85% are the E[1/5] type. Lactation resulted in an 88% reduction in total PI transcripts, of which the E[1/5] type was reduced 90% and the E[1/4/5] type 80%. By contrast lactation reduced the total levels of PII transcripts by only 50%. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an 8-fold increase in both E[1/4/5] and E[1/5] types when compared with explants prior to culture. PII transcripts, by contrast, were increased 2-fold by culture in insulin plus dexamethasone and this was entirely attributed to an increase in the expression of the E[2/4/5] type. Dexamethasone acts to potentiate the action of insulin on PI and PII transcript abundance and this effect is greatest for PI transcripts. This study has demonstrated that repression of the ACC-alpha gene in adipose tissue during lactation is largely achieved through attenuation of PI transcript abundance and may be related, in part, to a change in the sensitivity of the apparatus that regulates PI transcript steady-state levels to insulin.
Cyclin D3 in the mouse uterus is associated with the decidualization process during early pregnancy
Authors: Das SK , Lim H , Paria BC , Dey SK
Published: December 2004

In the mouse, the attachment reaction between the blastocyst trophectoderm and the receptive uterine luminal epithelium occurs at 2200-2300 h on day 4 of pregnancy and is rapidly followed by transformation of stromal cells into decidual cells (decidual cell reaction). This process can also be induced experimentally (deciduoma) by intraluminal oil infusion in the uterus on day 4 of pseudopregnancy. The decidual cell reaction is associated with up- and down-regulation of many genes in a cell-specific manner. Using mRNA differential display, we identified cyclin D3 as one of the genes that is upregulated in the uterus at the sites of blastocyst apposition during the attachment reaction. The levels of expression were low in the morning of days 1-4 as determined by Northern hybridization. In situ hybridization analysis showed that on days 1 and 2, signals were primarily localized in uterine epithelial cells, while signals were detected in both the stromal and epithelial cells on days 3 and 4. In contrast, with the initiation and progression of decidualization on days 5, 6 and 7, the levels of cyclin D3 mRNA were remarkably upregulated in stromal cells both at the mesometrial and the antimesometrial poles. However, on day 8, signals were primarily localized in stromal cells at the mesometrial decidual bed. Implanting blastocysts on these days also expressed cyclin D3 mRNA. In the progesterone-treated delayed implanting mice, the uterine levels of cyclin D3 mRNA were modest at the sites of blastocyst apposition, but were upregulated with the onset of implantation by estradiol-17beta. However, the decidual expression of cyclin D3 mRNA was not dependent on the presence of blastocysts, since increased expression also occurred in experimentally induced deciduoma in the absence of blastocysts. The importance of cyclin D3 in decidualization was further examined in Hoxa-10-deficient mice which show defective decidualization. The expression of cyclin D3 mRNA in Hoxa-10(-/-) uteri on day 5 was severely compromised after application of a deciduogenic stimulus on day 4 of pseudopregnancy. Collectively, the results suggest that cyclin D3 could be important for the process of decidualization.
Hormonal regulation of apolipoprotein AI
Authors: Hargrove GM , Junco A , Wong NC
Published: December 2004

Apolipoprotein AI (apo AI) is the major protein component of the serum high-density lipoprotein (HDL) particles. The antiatherogenic properties of apo AI alone or as part of HDL and their inverse correlation with the incidence of coronary heart disease underlie the clinical importance of the protein. A detailed understanding of the mechanisms by which apo AI is regulated will help us develop new and better ways to manipulate expression of the protein. Although there are many factors that influence apo AI expression, endogenous hormones are attractive because simple changes in abundance of these compounds will alter gene activity. Hormones belonging to the thyroid/steroid family that influence activity of the gene include thyroid hormone, glucocorticoids, gender-specific steroids and retinoic acid. Whereas thyroid, glucocorticoid and estradiol enhance activity of the gene, retinoic acid and androgens decrease it. The mechanisms that mediate the effects of the hormones include direct effects of the ligand and nuclear receptor complex on gene activity. However, indirect means involving the participation of transcription factors other than the hormone receptors are also possible. In summary, members of the same hormone family may have different mechanisms that mediate their activities on apo AI gene activity.
The structure and function of the ATP-sensitive K+ channel in insulin-secreting pancreatic beta-cells
Authors: Miki T , Nagashima K , Seino S
Published: December 2004

ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.
Gene expression of vasoactive intestinal contractor/endothelin-2 in ovary, uterus and embryo: comprehensive gene expression profiles of the endothelin ligand-receptor system revealed by semi-quantitative reverse transcription-polymerase chain reaction analysis in adult mouse tissues and during late embryonic development
Authors: Uchide T , Masuda H , Mitsui Y , Saida K
Published: December 2004

Vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2) is a 21 amino acid intestinal peptide characterized as a potent vasoactive and intestinal smooth muscle-contracting compound. To investigate the physiological roles of VIC/ET-2 further, we characterized the specificity of VIC gene expression relative to that of other members of the endothelin (ET) ligand-receptor system in adult mouse tissues and during embryonic development. Gene expression of ET-1, ET-3, ETA and ETB was ubiquitous in almost all tissues we examined while gene expression of VIC was localized to certain tissues. A high level of VIC gene expression was observed in ovary and uterus. The gene expression of VIC, relative to that of glyceraldehyde-3-phosphate dehydrogenase, was approximately 2.0%, 0.4%, and 2.3% in ovary, uterus, and intestine respectively, and was approximately 1.6 and 7. 1 times higher than that of ET-1 in ovary and intestine respectively. Thus, VIC may have some physiological role in adult ovary and uterus as well as intestine. In embryonic development, VIC gene expression sharply increased between 11 and 15 days post coitus and decreased after birth, suggesting an involvement in the later stages of embryonic development.
The beta-subunit of human chorionic gonadotrophin exists as a homodimer
Authors: Butler SA , Laidler P ,   Porter, Kicman AT , Chard T , Cowan DA , Iles RK
Published: December 2004

The free beta-subunit of human chorionic gonadotrophin (hCGbeta) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCGbeta may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo- and heterodimers, it is possible that the same may be true for hCGbeta. Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCGbeta contain hCGbetabeta homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCGbeta species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a |mf23 kDa monomer and a |mf46 kDa dimer were identified. Formation of hCGbetabeta homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCGbeta preparations in tumours.
Effect of growth hormone administration on IGF binding protein-3 mRNA levels in porcine tissues
Authors: Dunaiski V , Dunshea FR , Walton PE , Goddard C
Published: December 2004

The effect of short-term GH treatment on steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA levels in liver, kidney, longissimus dorsi muscle, stomach and jejunum was examined in pigs. Ten female crossbred pigs were allocated to either saline or GH (70 microg/kg/day) treatment by subcutaneous injection for 4 days. They were allowed to feed ad libitum, and were weighed daily. At the end of the treatment period, the pigs were slaughtered and samples of liver, kidney, skeletal muscle, stomach and jejunum were collected and total RNA was extracted. Steady-state levels of IGFBP-3 mRNA were quantified by RNase protection assay and were compared with the level of IGF-I class 1 and class 2 transcripts. IGFBP-3 mRNA increased in response to GH in both liver and kidney, but not in the other tissues sampled. Hepatic IGF-I mRNA responded to short-term GH treatment with a fourfold increase in IGF-I class 1 mRNA and an eightfold increase in IGF-I class 2 mRNA, which was liver specific. IGF-I class 1 mRNA was not responsive to GH treatment in other tissues. The short-term nature of this treatment suggests that the increase in hepatic IGFBP-3 and IGF-I transcripts is a relatively early response to treatment with GH, and that the increase in plasma concentrations of IGFBP-3 in response to GH are derived from the liver, the kidney, or both.
Expression of the vasotocin gene in the hypothalamus of intact and osmotically stimulated bullfrogs during metamorphosis
Authors: Hyodo S
Published: December 2004

To study the ontogeny of the vasotocin (VT) system and its contribution to anuran metamorphosis, VT mRNA levels were determined by Northern blot analysis in metamorphosing bullfrog tadpoles. Effects of osmotic stimulation on VT mRNA levels were also analyzed in order to follow the development of osmotic responsiveness of VT neurons. The intensity of hybridization signals for VT mRNA gradually increased during prometamorphic development. The increase became marked thereafter until metamorphic climax. Plasma osmolality and hematocrit remained unchanged before metamorphosis, and increased after metamorphic climax, indicating that climactic tadpoles in a semi-terrestrial environment were in a dehydrated condition. These increases correlated well with the increase in VT mRNA level. Immersion of tadpoles in 30% seawater (approximately 350 mOsmol) for 3 days increased plasma osmolality at all stages. No significant changes were observed in the VT mRNA level in response to this treatment during premetamorphic stages. The VT mRNA levels were significantly higher in the treated tadpoles after preclimax stages. Hyperosmotic treatment also increased hematocrit until early metamorphic climax, but did not alter it in tadpoles at late metamorphic climax. These results suggest that the responsiveness of VT-producing neurons to hyperosmotic or hypovolemic stimulation, or both, is established by the time of the metamorphic climax in bullfrog. The marked increase in VT mRNA levels at metamorphic climax stages of intact individuals is probably induced by dehydration. VT-stimulated water absorption and reabsorption in the target organs probably prevented the increase in hematocrit at late metamorphic climax. Thus VT may contribute importantly to osmoregulatory mechanisms in relation to adaptation to a semi-terrestrial habitat through the metamorphosis.
Selective activation of the probasin androgen-responsive region by steroid hormones
Authors: Kasper S , Rennie PS , Bruchovsky N , Lin L , Cheng H , Snoek R , Dahlman-Wright K , Gustafsson JA , Shiu RP , Sheppard PC , Matusik RJ
Published: December 2004

Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.
Increased expression of both short and long forms of prolactin receptor mRNA in hypothalamic nuclei of lactating rats
Authors: Pi XJ , Grattan DR
Published: December 2004

This study investigated expression of prolactin receptor (PRL-R) mRNA in selected hypothalamic nuclei of lactating rats (days 7-10 post partum) compared with dioestrous rats. Rat brains were frozen with liquid nitrogen and cut into coronal sections of 300 microm. From these sections, tissues were micropunched from the parietal cortex (CTX), choroid plexus (ChP), and five hypothalamic regions: supraoptic (SO), paraventricular (Pa), arcuate (Arc) and ventromedial hypothalamic (VMH) nuclei, and median eminence (ME). Expression of both short and long forms of PRL-R mRNA were evaluated by reverse transcription-PCR and Southern hybridisation. The results showed that the relative amount of short form mRNA in the ChP of lactating rats was significantly higher than in dioestrous rats. The short form of PRL-R mRNA was undetectable in the SO, Pa, VMH of dioestrous rats but was expressed at a significant level in lactating rats. Levels of long form mRNA in the ChP, SO, Pa and VMH in lactating rats were significantly increased compared with dioestrous rats. Moreover, the long form mRNA was induced in the CTX of lactating rats. In the Arc, levels of both forms of PRL-R mRNA tended to increase in lactating rats compared with dioestrous rats but changes were not statistically significant. Neither form of PRL-R mRNA was detectable in the ME in the two animal models. Increased expression of PRL-R mRNA in specific brain regions during lactation is consistent with the variety of PRL effects on the brain, and may help to explain profound physiological changes in the lactating mother.
The baboon: a model for the study of primate growth hormone receptor gene expression during development
Authors: Zogopoulos G , Nathanielsz P , Hendy GN , Goodyer CG
Published: December 2004

In subprimates, significant onset of growth hormone receptor (GHR) expression occurs only after birth whereas, in the human, GHR mRNA and protein are widely manifest from the first trimester of fetal life. Thus, it is likely that subprimates are not the best models for studying regulation of human GHR gene transcription, especially during early stages in development. Here we have explored the potential of the baboon as a more appropriate model. Baboon GHR cDNAs were cloned from postnatal liver by reverse transcription (RT)-PCR, using human GHR-specific primers. The encoded baboon GHR precursor protein has an identical signal peptide sequence to that of human and rhesus monkey GHRs, and the mature baboon GHR is also 620 amino acids long, with 95% and 98.5% amino acid identity to the human and rhesus monkey receptors respectively. Previous studies in the human have identified eight 5' untranslated region (5' UTR) variants of the GHR mRNA (V1 to V8, numbered according to their relative abundance). We cloned the baboon V1, V3 and V4 homologues by RT-PCR: these variants have a high degree (>92%) of sequence identity with their human counterparts and also diverge at an identical point, 12 nucleotides upstream of the start of translation. The expression pattern of these three GHR mRNA isoforms in baboon liver during development was characterized. Strong expression of baboon V1 and V4 was evident by 49 days of postnatal life (n=5, 49 days and adult (18.6-19.6 kg)); very low levels of V1, but not V4, were observed in younger animals (n=2, 6 and 30 days). In contrast, V3 5' UTR variant mRNA was present in all fetal (n=4, 141-155 days gestation) and postnatal (n=7, 6-19.6 days and adult (18.6 kg)) hepatic specimens examined. Analysis of postnatal kidney and lung (n=2, 19 and 19.6 kg) revealed that V3 transcripts are present in these tissues, but not V1 and V4. Together, these data demonstrate that, as in the human, baboon V1 and V4 expression is developmentally regulated and tissue specific, while the V3 isoform is more widely expressed. Therefore, it is likely that the regulatory regions of the baboon and human GHR genes are well conserved. Our findings suggest that the baboon is an appropriate animal model in which to define the mechanisms regulating GHR gene transcription during primate development.
In vitro expression of long and short ovine prolactin receptors: activation of Jak2/STAT5 pathway is not sufficient to account for prolactin signal transduction to the ovine beta-lactoglobulin gene promoter
Authors: Bignon C , Daniel N , Belair L , Djiane J
Published: December 2004

The recent finding that sheep had long (l-oPRLR) and short (s-oPRLR) prolactin receptors provided new tools to further explore prolactin signaling to target genes. Here we used CHO cells transfected with l-oPRLR or s-oPRLR cDNAs to compare the activation of known key steps of prolactin signaling by the two receptors. We found that prolactin stimulated l-oPRLR tyrosine phosphorylation, although it lacked the last tyrosine residue found in other long prolactin receptors. In addition, l-oPRLR and s-oPRLR both responded to prolactin stimulation by (1) Janus kinase 2 (Jak2) tyrosine phosphorylation, (2) DNA-binding activation of signal transducer and activator of transcription 5 (STAT5), (3) stimulation of transcription from a promoter made of six repeats of STAT5-responsive sequence. However, although it contains STAT5-binding consensus sequences, the ovine beta-lactoglobulin promoter (-4000 to +40) was transactivated by l-oPRLR, but not by s-oPRLR. Taken together, our results indicate that activation of Jak2/STAT5 pathway alone is not sufficient to account for prolactin-induced transcription of this milk protein gene, and that sequences of its promoter, other than STAT5-specific sequences, account for the opposite transcriptional activation capabilities of l-oPRLR and s-oPRLR.
Oestrogens regulate pituitary alpha2,3-sialyltransferase messenger ribonucleic acid levels in the female rat
Authors: Damian-Matsumura P , Zaga V , Maldonado A , Sanchez-Hernandez C , Timossi C , Ulloa-Aguirre A
Published: December 2004

Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gland in multiple molecular forms. Increased acidic/sialylated FSH charge isoforms are associated with conditions characterized by a low oestrogen output. In the present study, we analysed the dynamics of the changes in mRNA levels of the enzyme Galbeta1,3[4]GlcNAc alpha2,3-sialyltransferase (2,3-STase) (one of the enzymes that incorporate sialic acid residues into the FSH molecule) in intact and ovariectomized rats. The anterior pituitaries of 4-day regularly cyclic adult female Wistar rats were obtained at 1000 h on the days of pro-oestrus (P), oestrus (O), dioestrus 1 (D1) and dioestrus 2 (D2), at 0200 h, 1400 h, 1800 h and 2200 h on D1, at 1800 h on day of O and at 1000 h after 7, 14, 21, 28 and 45 days of oophorectomy performed on the morning of P. Total RNA was isolated from each gland and the 2,3-STase levels were measured by Northern blot hybridization analysis employing a 346-base pair cDNA probe encoding for a non-conserved amino acid sequence of the catalytic domain of the enzyme. Maximal levels of the enzyme mRNA were detected at 1000 h on D1; thereafter, they progressively decreased by 60% during the ensuing 24 h, reaching the lowest concentration values (26% of the maximally observed level on D1) at 1000 h on day of P and remaining unchanged during the morning of O. Administration of the potent oestradiol receptor antagonist ICI 182,780 at 1000 h on D1 completely reverted the time-dependent decrease in 2,3-STase mRNA levels observed during the afternoon of D1, whereas oestradiol benzoate administered at 1000 h on day of O significantly reduced the enzyme mRNA levels (to 21% of the levels detected in vehicle-treated controls). In ovariectomized rats, the alpha2,3-STase mRNA progressively increased from day 21 to day 45 post castration. Administration of oestradiol benzoate on day 28 after oophorectomy significantly reduced the 2,3-STase mRNA levels (to 36% of the levels detected in vehicle-injected controls); ICI 182,780 partially counteracted this oestradiol-mediated effect. The dynamics of these changes in 2,3-STase mRNA levels partially correlated with changes in the relative abundance of the FSH charge isoforms separated by preparative chromatofocusing of anterior pituitary extracts, particularly in glands obtained during the morning of P and O. These data demonstrate for the first time that pituitary 2,3-STase is a hormonally-regulated enzyme and that the changes in transcription and/or stability of its mRNA may be involved, in part, in the post-translational processing of the FSH molecule during certain physiological conditions.
Testosterone effect on growth and growth mediators of the GH-IGF-I axis in the liver and epiphyseal growth plate of juvenile rats
Authors: Zung A , Phillip M , Chalew SA , Palese T , Kowarski AA , Zadik Z
Published: December 2004

Several studies have suggested that testosterone may have a direct, GH-independent effect on growth. In order to assess possible mechanism(s) whereby testosterone exerts its growth-promoting effect, we evaluated its effect on growth mediators of the GH-IGF-I axis, in both the liver and the epiphyseal growth plate (EGP). Testosterone was administered to peripubertal rats and the responses of mRNA of GH receptor, IGF-I, IGF-I receptor and IGF-binding proteins-1 and -3 (IGFBP-1 and IGFBP-3) as well as circulating IGF-I were evaluated in two time-related models: over 12 h after a single injection (short-term study) and 10 days after continuous administration (long-term study). Rats in the short-term study were castrated and were killed 1, 4, 6 and 12 h post injection. Rats in the long-term study were divided into two groups: castrated vs castrated and hypophysectomized, in order to assess the effect of testosterone in the presence and absence of GH. mRNA levels were determined by RNase protection assay, and serum IGF-I by RIA. Testosterone enhanced weight gain in the rats treated for 10 days, a change that was similar in the presence or absence of GH. This effect was relatively small, however, by comparison with the total weight gained without testosterone. Testosterone had no effect on hepatic IGF-I mRNA abundance but induced a reduction in circulating IGF-I levels, in both the short- and long-term study. Testosterone had no effect on hepatic GH receptor and IGFBP-3 mRNA levels but resulted in a transient, short-term elevation in IGFBP-1 mRNA levels that was maximal 4 h post injection.In the EGP, neither testosterone administration nor hypophysectomy had any effect on IGF-I and IGF-I receptor mRNA levels. However, testosterone increased GH receptor mRNA abundance after 10 days of continuous administration in hypophysectomized rats only.These data suggest that the effect of testosterone on growth (as assessed by weight gain) is small and is not mediated by changes in hepatic gene expression of IGF-I, IGF-I receptor, IGFBP-1, IGFBP-3 or circulating IGF-I. At the EGP, the testosterone effect on linear growth is not mediated through changes in mRNA abundance of IGF-I and IGF-I receptor. The small but significant elevation of GH receptor mRNA levels in hypophysectomized rats may suggest a testosterone-mediated augmentation of a GH effect at the target organ.
Resolution and identification of Gq/G11alpha and Gialpha/Goalpha proteins in brown adipose tissue: effect of cold acclimation
Authors: Bourova L , Novotny J , Svoboda P
Published: December 2004

Levels of guanine nucleotide-binding proteins G(q)alpha and G(11)alpha, which produce receptor regulation of phospholipase C, were measured immunologically in purified plasma membrane fractions of hamster brown adipose tissue (BAT). This was achieved by immunoblotting with antisera (CQ series) that identify these two polypeptides equally, following separation of the plasma membranes using SDS-PAGE in the presence of 6 M urea, i.e. conditions that can resolve G(q)alpha and G(11)alpha. The ratio of levels of G(q)alpha to G(11)alpha was 1:1. A similar approach was used for resolution and identification of G(o)1alpha and G(o)2alpha, the latter representing the prevailing form of G(o)alpha proteins in this tissue. Although clearly recognized in brain microsomes, which were used as positive controls, no detected levels of G(o)*alpha protein were noted. Using specific anti-peptide antibodies directed against the carboxy-terminal decapeptide of G(i)3alpha, this G protein was also found to be expressed in BAT tissue. Cold acclimation resulted in reduction of the plasma membrane levels of all these Galpha proteins.
Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain
Authors: Thomas JL , Evans BW , Blanco G , Mason JI , Strickler RC
Published: December 2004

Human 3beta-hydroxysteroid dehydrogenase/steroid Delta(5)-Delta(4)-isomerase (3beta-HSD/isomerase) is a bifunctional, single enzyme protein that is membrane-bound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3beta-HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3beta-HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3beta-HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3beta-HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH(2)-terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/quaternary structure of this key steroidogenic enzyme.
Complex mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-II) and IGF-binding protein-2
Authors: Badinga L , Song S , Simmen RC , Clarke JB , Clemmons DR , Simmen FA
Published: December 2004

The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.
Structural and functional analysis of the promoter of a mouse gene encoding an androgen-regulated protein (MSVSP99)
Authors: Brochard D , Morel L , Veyssiere G , Jean C
Published: December 2004

MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is a member of the rat and mouse seminal vesicle secretory protein family. The gene encoding MSVSP99 is under androgenic control and we demonstrate here that this regulation involves a complex interplay of positive and negative regions. First, we show that the promoter region (-387/+16) sufficient to mediate a full androgen induction is a complex enhancer organized in two regulatory regions. These two regions are inactive individually and must act together to confer a 40-fold androgen induction to the MSVSP99 gene and androgen responsiveness is not only dependent on the presence of functional androgen response element (ARE) sequences but results from complex cooperations between ARE and non-ARE sequences forming an androgen response unit. Secondly, we characterized a new regulatory region (-824/-632) that decreases androgen-dependent transcriptional activity of the MSVSP99 promoter. This region, also able to repress the transcriptional activity of the heterologous thymidine kinase promoter, contains a functional promoter on the inverted strand (-826 to -387) and we identified a transcription initiation site located at position -639 with respect to the cap site of the MSVSP99 promoter. Sequence analysis of the flanking DNA also revealed that the MSVSP99 gene is surrounded by long interspersed repeated sequences called LINEs.
Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems
Authors: Beattie J , Borromeo V , Bramani S , Secchi C , Baumbach WR , Mockridge J
Published: December 2004

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.