Growth hormone-binding protein in the goat: characterization, evolution under exogenous growth hormone treatment, and correlation with liver growth hormone receptor levels

This report describes the identification and characterization of a specific, high-affinity growth hormone-binding protein (GHBP) in lactating goat serum. Serum samples were incubated with [¹²⁵I]human GH as ligand and in the absence or in the presence of bovine GH as competitor. GH-GHBP complex formation was performed by high-performance liquid chromatography, and the radioactivity was recorded on-line with a Berthold LB detector connected to a computer. The results showed that a serum protein was able to bind specifically to human GH and bovine GH but not to ovine prolactin. Scatchard plots indicated an affinity constant of 4.5 × 10⁸ M⁻¹ and a maximum binding capacity of 4.8 × 10⁻¹⁰ mol/l. In addition, we conducted a 4-wk study to determine the effects of recombinant bovine GH administration on milk production in lactating goats. The effects of recombinant bovine GH treatment on milk production and on the regulation of GHBP and hepatic GH receptor levels were studied. As expected, recombinant bovine GH injected daily increased yields of milk, fat, protein (40, 61, and 40%, respectively), and circulating insulin-like growth factor 1 concentrations compared with controls. During the pretreatment and treatment periods, the control goats exhibited a constant amount of GHBP in serum. No consistent effect of GH treatment on GHBP level was observed. The binding of [¹²⁵I]bovine GH to hepatic microsomal membranes of GH-treated goats was significantly decreased compared with that of control goats. After MgCl₂ desaturation of membranes, the results demonstrated that the down-regulation of GH hepatic receptors, observed for the treated goat group, was induced by receptor occupancy without modification of binding affinity. The GH receptor gene expression, analyzed by slot blot and hybridization with an [-³²P]GH receptor cDNA probe, was not modified by the GH treatment. In lactating goats, the galactopoietic effect of exogenous GH involved a hepatic receptor occupancy. The individual concentration of GHBP in serum cannot explain the individual variations of responses to GH treatment in goats.     

Ontogeny of a specific high-affinity binding site for ovine placental lactogen in fetal and postnatal liver

Ovine placental lactogen (oPL) exerts actions in sheep and rodent fetal tissues that growth hormone (GH) does not. However, in postnatal tissues, both oPL and GH possess these activities. Although a high-affinity binding site for oPL in ovine fetal liver has been reported, some investigators believe this to be the GH receptor. It was our objective to discriminate between oPL and GH binding to fetal liver microsomes using competitive saturation analyses. Microsomal membranes from fetal liver (Days 60, 90, 105, 120, and 135 of gestation) and postnatal liver (1 wk of age) were incubated with increasing amounts of [¹²⁵I]oPL in the absence or presence of a 100-fold molar excess of unlabeled oPL. Saturable binding of [¹²⁵I]oPL was observed with fetal liver and postnatal liver microsomes. The K_d of the oPL-binding site in fetal liver was 122.1 ± 8.2 pM (mean ± standard error), and receptor concentrations remained relatively constant (9.8 ± 1.1 fmol/mg of membrane protein) across gestation. The highest concentration of oPL binding was detected in 1-wk postnatal liver microsomes (53.0 fmol/mg of membrane protein). Saturation analyses using [¹²⁵I]GH and [¹²⁵I] prolactin (PRL) were also conducted with fetal liver membrane preparations. Although specific binding for these two radiolabeled ligands was observed in control tissues, no specific binding was observed in fetal liver. These data are in agreement with earlier reports that a high-affinity binding site for oPL exists in fetal tissues. The fact that saturable binding could not be demonstrated for either GH or PRL with fetal liver microsomes contradicts recent suggestions that oPL is binding the GH receptor.     

Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration

Postweaning serum insulin-like growth factor-I (IGF-I) concentrations and serum IGF binding proteins (IGFBP) were investigated in 68 (1992 Fall-born) and 84 (1999 Fall-born) Angus cattle selected for either high or low serum IGF-I concentrations since 1989. Relative serum levels of IGFBP were determined by [¹²⁵I]IGF-I Western ligand blotting. IGFBP species of 38–42, 34, 30, and 24 kDa were identified. The 34 kDa species was identified as IGFBP-2 by immunoblot analysis. No significant line effects were observed for any of the IGFBP. In both 1992 and 1999, heifers had higher IGFBP-2 levels than bulls (P<0.0005). In 1992 calves, relative levels of the 38–42 and 24 kDa species were significantly correlated with serum IGF-I concentration. In 1999 calves, none of the IGFBP were correlated with serum IGF-I, although IGFBP-2 was negatively correlated with several measures of body weight. No significant line effects were observed for growth or serum IGF-I traits in 1992 calves. However, 1999 high line calves had higher serum IGF-I concentrations and body weights than low line calves (P<0.05). In both 1992 and 1999 calves, bulls had higher serum IGF-I concentrations and body weights than heifers (P<0.05). Thus, while selection for high versus low serum IGF-I concentrations has resulted in divergence between the selection lines and also in changes in body weights, it has not resulted in changes in serum IGFBP levels. Furthermore, circulating IGFBP-2 appears to be higher in heifers than in bulls, and also appears to be negatively correlated with body weights.     

Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells.

Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.     

Colostral and milk insulin-like growth factors and related substances: mammary gland and neonatal (intestinal and systemic) targets

The identification of hormones and regulatory factors in colostrum and milk has led to intensive investigations on their roles in the development and maintenance of the mammary and neonatal tissues. Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in transgenic mice influence mammary biology gland towards the end of lactation. In the bovine, IGFBP-3 is the major IGFBP in mammary secretions. In addition to binding IGFs, IGFBP-3 also binds to lactoferrin (Lf). Secreted IGFBP-3 re-enters mammary epithelial cells and with the presence of a nuclear localization sequence, IGFBP-3 and Lf enter the nucleus. Nuclear IGFBP-3 affects apoptotic signaling through the retinoic-x-receptors, while Lf affects apoptotic events through unknown mechanisms. Such interactions likely influence mammary development and involution. Furthermore, ingested colostral bioactive factors can exert regulatory functions in neonates. Intestinal receptors for IGFs and insulin are modified by age and/or diet. Feeding IGF-I had no effect, but colostrum extracts had small intestinal effects (stimulation of proliferation and villus size), suggesting that several factors, rather than one single bioactive factor were responsible. Systemic changes of metabolic and endocrine profiles in neonates depend on composition, amounts, time and duration of feeding colostrum. Early postnatal colostrum intake is not only important for the provision and absorption of immunoglobulins. Thus, in neonatal calves the lack of colostrum intake during the first 24h after birth results in a low immunoglobulin G, beta-carotene and Vitamin A status that persists for weeks and plasma patterns of fatty acids, essential amino acids and the glutamine/glutamate ratios are affected. In calves oral administration of IGF-I had no and feeding of colostrum whey extracts had only minor effects on metabolic and endocrine traits. Thus, mammary secretions influence regulatory functions of mammary and neonatal tissues.     

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum.

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [(125)I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL (p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner (p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production (p < 0.05). Both IGFBP-2 and -3 inhibited [(125)I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.     

Effect of bovine somatotropin and food deprivation on β-adrenergic and A1 adenosine receptor binding in adipose tissue of lactating cows

Lactating Holstein cows were used to assess the effect of bovine somatotropin (bST; n = 8) and fasting (FAST; n = 4) on ligand binding to β-adrenergic (BAR) and TYP e-1 adenosine (A₁R) receptors in adipose tissue. Cows received exogenous bST (sometribove; 40 mg/d) or no hormone (control) for 4 d in a single-reversal design with a 7-d interval between treatment periods. Subcutaneous adipose tissue biopsies were taken on day 4 of each treatment. Eight d after the bST regimen, 4 cows were fasted for 3 d and adipose biopsies were taken. Ligand binding was quantified with a postnuclear, total adipose tissue membrane preparation (100,000 × g pellet). Binding to BAR and A₁R was assessed with the antagonists [¹²⁵I]iodocyanopindolol (ICP) and [³H]8-cyclopentyl-1,3-dipropylxanthine (DCPCX), respectively. The binding affinity (K_d) of BAR for ICP was not affected by bST but was enhanced by FAST; maximal binding (B_max) was increased with bST treatment (P < 0.06) and reduced by FAST (61%, P < 0.01). K_d values for DCPCX binding to A₁R were not changed by bST or FAST. bST did not affect B_max for A₁R; however, FAST reduced the B_max by 38%. Data highlight the differential regulation of BAR and A₁R by bST and FAST.     

Lactoferrin interaction with retinoid signaling: cell growth and apoptosis in mammary cells

Lactoferrin (Lf) is a multifunctional iron-binding protein that was first identified in mammary secretions, but is synthesized by most mammalian tissues. The protein has a signal sequence that dictates secretion; it also has a nuclear localization sequence that facilitates entry into the cell nucleus. The mechanism of the latter action is currently unknown, but is thought to occur via a Lf receptor. Lactoferrin content of mammary tissue and secretions varies with developmental state; it is synthesized in mammary tissue at high levels during both pregnancy and involution, and during mammary infections. Using fluorescent (FITC)-labeled holo-bLf, we show that bovine primary epithelial cells and MCF-7 breast cancer cells do not translocate the exogenously added Lf to the nucleus after culture in serum free media (SFM). However, the supplementation of SFM with 1 μM all-trans retinoic acid (atRA) caused breast cancer cells to gain the capacity to take up labeled bLf into the cell nucleus. Primary bovine mammary cells (MeBo) exhibited similar capacity in culture. This suggests that in addition to Lf, one or more components modulated by atRA, are necessary for nuclear translocation to occur. Transfection experiments with atRA treated MCF-7 cells containing retinoic acid response element reporter constructs showed that the extracellular application of lactoferrin alters reporter gene expression. Lactoferrin increased a DR5 luciferase response element in a dose-dependent manner only when atRA was applied. Immunocytochemical markers for the cell cycle (Ki67) and apoptotic events (Caspase-3 and PARP-85) showed that lactoferrin alters the atRA-induced phenotype, blocking apoptosis and maintaining cell cycle activity in both MCF-7 and MeBo cells in the presence of 1 μM atRA. We propose that nuclear lactoferrin interacts with retinoic acid signaling pathways in cells and alters/blocks the signals so that cells remain in the cell cycle and/or do not enter the apoptotic pathway.     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

Measurement of dimeric inhibin in porcine serum: Evidence for low concentrations and existence of binding proteins

An immunoradiometric assay and serum extraction procedure were developed to measure dimeric inhibin in porcine serum with minimal interference by putative inhibin-binding proteins. Assay sensitivity was 50 pg/tube, and it incorporated antibodies against the N-terminal region of inhibin's -subunit, -(1–25)-Ab, and against the C-terminal region of inhibin's β_A-subunit. To determine whether inhibin-binding proteins were present in porcine serum, serum was incubated with [¹²⁵I]-recombinant human (rh)-inhibin and then chromatographed by gel filtration. Radioiodinated rh-inhibin was associated with protein(s) >600 kDa. Radioiodinated rh-inhibin also was incubated with ₂-macroglobulin, an inhibin-binding protein in human and rat serum. Elution profiles were similar for serum and ₂-macroglobulin. Serum- and ₂-macroglobulin-[¹²⁵I]rh-inhibin complexes dissociated upon exposure to 8 M urea. Porcine serum was treated with urea, after which inhibin was isolated and concentrated. The recovery of rh-inhibin added to starting serum was 28%. Concentrations of endogenous dimeric inhibin were <28 pg/ml in serum collected from sows at random stages of the estrous cycle and were <21 pg/ml in serum collected from sows 2 d postweaning. Results demonstrate that 1) concentrations of dimeric inhibin are low in porcine serum, and 2) an inhibin-binding protein(s), consistent with ₂-macroglobulin, is present in porcine serum.     

Insulin binding to bovine mammary membranes: comparison of microsomes versus smooth membranes

The study was undertaken to determine if membrane preparations of bovine mammary tissue bound insulin. If binding occurred, it was also the intent to compare binding kinetics between microsomes and smooth membranes. Insulin binding to bovine mammary membranes attained equilibrium, was saturable and was specific for insulin. Additional studies showed binding to be pH sensitive and maximal at 10 mM calcium. Binding affinity of insulin to microsomes and smooth membranes was similar, with the exception that smooth membranes bound 1.8 times more insulin per unit of membrane protein than microsomes. Two different methods were used to generate data for kinetic analysis of the insulin-receptor interaction in microsomes. Competitive binding assays (.6 ng [125I]insulin plus 0 to 100 ng insulin) indicated the presence of two binding sites with dissociation constants (Kd) of .32 and 15.8 nM. Direct titration of microsomes with [125I]insulin (.02 to 10 ng/ml) revealed two binding sites with Kd of .017 and .31 nM. The affinity of the second binding site measured by the competitive binding assay method (Kd of 15.8 nM) is low and therefore may not be of physiological importance for insulin action. Insulin appears to bind to two high-affinity receptor sites in bovine mammary microsomes with Kds of .017 nM and .32 nM. These findings show that bovine mammary tissue contains receptors for insulin. In addition, isolation of smooth membranes from microsomes enriches the number of insulin receptors per unit of membrane protein without altering their binding characteristics.     

Blocking of nonspecific IgG in indirect radioimmunoassay for detecting Ascaris suum antigens

The sensitivity of an indirect radioimmunoassay (IRIA) used for detecting larval body wall (LBW) antigens of Ascaris suum was enhanced by using various blocking agents which prevented nonspecific binding of immunoglobulin G (IgG) or free ¹²⁵I without preventing binding of specific antibodies to the antigen. The use of blocking agents reduced counts for both positive and negative sera, resulting in an increase in calculated binding ratios (BR) and deltas and, thus, in the sensitivity of the assay. The relative effectiveness of blocking agents, in decreasing order, were turkey serum (TS), rabbit gamma globulin (RGG), rabbit whole serum (RS), bovine serum albumin (BSA), rabbit IgG (RIgG), bovine alpha globulin (BAG), bovine gamma globulin (BGG) and Tween-20.     

The mRNA expression of the members of the IGF-system in bovine corpus luteum during induced luteolysis

The components of the IGF-system were shown to be differentially regulated in bovine antral follicles and corpora lutea (CL) during different stages of the estrous cycle, and to have important functions for specific stages. The aim of this study was to investigate the detailed pattern of mRNA expression of most constituents of the IGF-system and their possible involvement in prostaglandin (PG)F2-induced luteolysis in the bovine CL. Therefore, cows in the mid-luteal phase (days 8–12) were injected with the PGF2-analogue Cloprostenol, and CL were collected by transvaginal ovariectomy at 2, 4, 12, 48 and 64 h after PGF2-injection. Real-time RT-PCR using SYBR Green I detection was employed to determine mRNA expressions of the following factors: ubiquitin (UBQ), insulin-like growth factor I (IGF I), IGF II, IGF-receptor type 1 (IGFR-1), growth hormone receptor (GH-R) and IGF-binding proteins-1–6 (IGFBP-1–6). Total extractable RNA decreased with ongoing luteolysis. IGFBP-1 mRNA was significantly up-regulated at 2 h after PGF2 and maximal at 4 h with a 34-fold increase. IGFBP-5 mRNA was significantly up-regulated after 12 h with a maximum of an 11-fold increase at 64 h. For GH-R, IGFR-1, IGF II, IGFBP-3 and -4 mRNA expression, we found a significant down-regulation in certain stages. There was a significant up-regulation for IGFBP-2 and -6 mRNA at 64 h after induced luteolysis. There were no significant changes in IGF I mRNA expression. In conclusion, the IGF-system with all its components seems to play an important role in the very complex process of PGF2-induced luteolysis in bovine CL.     

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

The role of IGFBP-5 in mammary gland development and involution

Insulin-like growth factor-I (IGF-I) plays an important role as a survival factor during mammary gland development and remodelling during involution of the mature/lactating mammary gland, and elevated concentrations have been associated with increased risk of breast cancer. The actions of IGF-I are modulated by a family of binding proteins (IGFBPs) and we have shown that IGFBP-5 is associated with cell death in the mammary gland and more recently provided the first evidence that it is causally related to apoptosis of the mammary gland. A transgenic mouse expressing IGFBP-5 on a mammary-specific promoter led to impaired mammary development involving inhibition of IGF-signalling and involving members of the Bcl-2 family. Subsequent studies in vitro and in vivo using exogenous IGFBP-5 treatment have added support to this concept. Although the effects of IGFBP-5 did appear to involve inhibition of IGF action, a role for IGF-independent effects cannot be ruled out. Such IGF-independent effects involve potential interactions with components of the extracellular matrix involved in tissue remodelling including plasminogen activator inhibitor-1 (PAI-1). In addition, intracellular events involving nuclear localisation of IGFBP-5 have been shown to have the ability to inhibit cell proliferation. Thus, IGFBP-5 seems important for regulating both apoptosis and cell proliferation in the mammary gland during development and post-lactation involution.     

A local increase in the mammary IGF-1: IGFBP-3 ratio mediates the mammogenic effects of estrogen and growth hormone

A single epithelium-free mammary fat pad was surgically prepared in each of twenty-five one-month-old, Friesian heifers. At 18 mo of age, heifers were randomly assigned to one of four treatment groups. Treatments were: control (C), growth hormone (GH), estrogen (E) or growth hormone + estrogen (GE). Hormones were administered for 40 hr before the animals were sacrificed to provide mammary samples of parenchyma (PAR), intact fat pad (MFP), and epithelium-free or "cleared" fat pad (CFP). IGF-1 and IGF binding protein-3 (IGFBP-3) mRNA was highest in CFP and MFP whereas the protein products were highest in PAR. IGFBP-2, a 28-kDa IGFBP and a 24-kDa IGFBP were more abundant in CFP and MFP. E and GH increased incorporation of [(3)H]thymidine into DNA of PAR. Incorporation of [(3)H]thymidine into the DNA of MFP or CFP was minimal. Coincident with the changes observed in mammary epithelial proliferation, E increased IGF-1 protein in MFP and PAR, and to a lesser extent in CFP. E tended to increase IGF-1 mRNA levels in MFP, but not CFP implying that the regulation of IGF-1 expression is modulated by adjacent epithelium. GH and E reduced IGFBP-3 protein in PAR and increased the 24-kDa IGFBP in CFP and MFP. Increased proliferation of mammary parenchymal cells was associated with increased IGF-1 and reduced IGFBP-3 protein in mammary tissue. An increase in the ratio of mammary IGF-1: IGFBP-3 likely increases the proportion of the mammary IGF-1 available to stimulate proliferation. These data also indicate that stromal: epithelial interactions regulate the IGF-1 axis in mammary tissue.     

Ontogeny of hepatic bovine growth hormone receptors in cattle

A series of studies examined the binding characteristics and ontogeny of hepatic growth hormone binding sites in dairy bulls on d 2, 30, 180, and 365 of age. Binding of iodinated recombinant bovine growth hormone ([125I]rbGH) to liver membrane receptors was membrane protein-dependent. Receptors were considered growth hormone-specific, because physiological concentrations of bovine prolactin (bPRL) failed to displace [125I]rbGH from bovine hepatocyte membranes. Only 50% of [125I]rbGH was bound reversibly to hepatic microsomes. Addition of dithiothreitol (DTT) to the receptor-assay buffer increased the binding of [125I]rbGH to hepatic membranes in a time-dependent manner. Moderate concentrations of Ca++ and Mg++ in the receptor-assay buffer had no detectable effects on binding of [125I]rbGH to hepatic microsomes. In growing dairy bulls, specific binding of [125I]rbGH per milligram of membrane protein increased from 1.9 +/- 1.8% at d 2 to 14.1 +/- 1.8% at d 180 and then declined to 5.2 +/- 1.6% at d 365. Likewise, concentration of insulin-like growth factor (IGF)-I in serum was low during the 1st mo of age (d 2, 13.3 +/- 8.8 ng/ml; d 30, 9.7 +/- 8.8 ng/ml), but it became maximal at d 180 (151.0 +/- 8.8 ng/ml). Circulating concentrations of IGF-II increased linearly during the 1st yr of growth. Serum concentrations of GH, triiodothyronine, and thyroxine declined from 39.9 +/- 6.5, 2.7 +/- .2, and 75.4 +/- 4.6 ng/ml at d 2 to 16.5 +/- 6.5, 1.3 +/- .2, and 53.4 +/- 4.6 ng/ml at d 30, respectively, and remained low through 1 yr of age. Insulin concentration in serum did not change significantly with development. Results indicated that increasing concentrations of specific bGH receptors in the bovine liver may play a key role in regulating postnatal growth in cattle.     

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.     

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus)

Objective To examine circulating total and free thyroid hormone (T₃ and T₄) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas.
Design A prospective study.
Procedure Koala serum T₃ and T₄ were measured by radioimmunoassay. T₄ binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay.
Results Mean total T₄ concentrations were 3.2 ± 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T₄ was 3.3 ± 2.1 pM. Total and free T₃ were 0.4 ± 0.2 nM and 1.4 ± 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [¹²⁵I]-T₄-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T₄ binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L.
Conclusion These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.