Effect of dietary energy and somatotropin on components of the somatotropic axis in Holstein heifers

The somatotropic axis, consisting of growth hormone (GH), GH receptor (GHR), insulin-like growth factor (IGF)-I, IGF binding proteins (IGFBP), and IGF receptors, controls growth and mammary development in heifers. Manipulation of the axis with recombinant bovine somatotropin (rbST) improves heifer growth and reduces age at first calving. The effects of rbST are influenced by dietary energy through partially understood mechanisms. The objective was to characterize the somatotropic axis in Holstein heifers fed a diet for either low or high rate of gain and treated with or without rbST. Heifers (120 d of age) were assigned to one of 2 diets to gain either 0.8 kg/d (low, n = 18) or 1.2 kg/d (high, n = 20). Within each diet, half of the heifers (n = 9 for low and n = 10 for high) received daily rbST injections (25 microg/kg of body weight). Treatments and diets continued until slaughter (2 mo after puberty). Blood was collected 2x per week, and a frequent sampling window was performed 1 d before slaughter. Liver was collected at slaughter. Feeding a high diet or treating with rbST increased serum IGF-I and decreased serum IGFBP-2. The observed changes in serum IGF-I and IGFBP-2 were reflected in their respective liver mRNA amounts. Feeding a high diet decreased serum GH concentrations after rbST injection, but the stimulatory effect of rbST on serum IGF-I was nonetheless greater in high-diet heifers. The differential IGF-I response may be explained by greater GHR 1A in the liver of high-diet heifers. We conclude that a high-gain diet modifies the somatotropic axis in rbST-treated heifers by decreasing serum GH but increasing serum IGF-I after rbST treatment. Greater IGF-I (indicative of an increased GH response) may be a consequence of greater GHR 1A expression in the liver.     

Effect of negative energy balance on the insulin-like growth factor system in pre-recruitment ovarian follicles of post partum dairy cows

Post partum negative energy balance (NEB) in dairy cattle is associated with a delayed return to ovarian cyclicity and reduced fertility. This study compared the IGF system of pre-recruitment ovarian follicles between cows in mild (n = 6) or severe (n = 6) NEB during early lactation. Ovaries were collected in the second week post partum, when circulating concentrations of IGF-I and glucose were lower (P < 0.01) in severe NEB cows. mRNA expression for IGF-II, type 1 IGF receptor (IGF-1R) and IGF-binding proteins (IGFBP)-1 to IGFBP-6 was determined by in situ hybridisation in individual follicles using radiolabelled oligonucleotide probes. Follicles were classified as very small (1-2.5 mm) or small (2.5-5 mm) and healthy or atretic. Relative mRNA concentrations were measured as optical density (OD) units using image analysis. Thecal IGF-II mRNA expression was highest in very small, healthy follicles (P < 0.05). Granulosa cell IGFBP-2 was the only component to change with EB status, with higher mRNA expression in mild compared with severe NEB cows (P < 0.05). IGFBP-1 and IGFBP-3 mRNA expression were undetectable. IGF-1R, IGFBP-4 and IGFBP-5 mRNA expression were not significantly altered by follicle size or health, but IGFBP-5 tended to increase in atretic follicles. The pattern of IGFBP-6 mRNA expression in theca paralleled that of IGF-II mRNA, with higher (P < 0.05) levels in healthy, very small follicles. In conclusion, the reduced expression of IGFBP-2 mRNA in severe NEB cows may alter the bioavailability of circulating IGF-I and locally produced IGF-II to modulate the pre-recruitment stages of follicles required to maintain normal post partum ovarian cyclicity.     

Effect of long-term infusion with recombinant growth hormone-releasing factor and recombinant bovine somatotropin on development and function of dominant follicles and corpora lutea in Holstein cows.

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.     

Negative energy balance in dairy cows is associated with specific changes in IGF-binding protein expression in the oviduct

Negative energy balance (NEB) during early lactation in dairy cows leads to an altered metabolic state that has major effects on the production of IGF family members. Low IGF-I concentrations are associated with poor fertility and therefore we aimed to determine whether NEB exerts a direct effect on IGF expression in the postpartum oviduct. Multiparous Holstein cows were allocated to two treatments (each n=6) designed using differential feeding and milking regimes to produce either mild NEB (MNEB) or severe NEB (SNEB). Animals were slaughtered in week 2 of lactation when divergent metabolic profiles were evident. Oviducts were collected for RNA analysis by real-time RT-PCR and in situ hybridisation. Quantitative measures in oviduct gene expression were obtained for all members of the IGF family (IGF-I/II, IGF-binding proteins (IGFBP) 1-6 and receptors for IGF types 1 and 2), insulin A/B, GH, glucocorticoid and oestrogen alpha/beta. Expression of IGFBP-2 and IGFBP-6 (both of which have a high affinity for IGF-II) was decreased in SNEB relative to MNEB (P<0.05). No other gene was altered by NEB, but IGF-II, IGFBP-3, IGFBP-5 and IGFBP-6 all showed differential expression in different regions of the oviduct. These results indicate that, in addition to low circulating IGF-I after calving, NEB may also influence IGF availability in the oviduct indirectly through changes in specific IGFBP expression. It is possible that the predicted increased signalling by IGF-II may perturb embryo development, contributing to the high rates of embryonic mortality in dairy cows.     

Effect of nitric oxide on the expression of insulin-like growth factors and the insulin-like growth factor binding proteins throughout the lifespan of the human corpus luteum

The presence of insulin-like growth factors (IGF), IGF binding proteins (IGFBP) and IGF receptor type 1 (IGF-IR) in the human corpus luteum was investigated by examining the expression and production of related proteins throughout the lifespan of the corpus luteum and the action of nitric oxide upon their production. The expression of proteins in corpora lutea from the early, mid-and late luteal phases was assessed by immunohisto-chemistry, evaluated by a semi-quantitative analysis and the functional study was performed in corpus luteum explants incubated with nitric oxide donors. IGF-I and -II and IGFBP-1 and -3 were measured in the culture media by specific immunoassays. The results showed that IGF-I and -II, IGFBP-1 to -6 and IGF-IR were detected in the human corpus luteum throughout the luteal phase. Moreover, the expression and production of IGF-I and IGFBP-1 increased progressively from corpora lutea from the early to late luteal phases (P < 0.05), whereas the expression and production of IGFBP-2, -4 and -5 were significantly higher in corpora lutea from the mid-luteal phase (P < 0.05). No differences were observed in the expression of IGF-II, IGFBP-3 and -6 and IGF-IR throughout the lifespan of the corpus luteum. However, functional studies showed that nitric oxide donors elicited a stimulatory action on production of IGF-I in corpora lutea from the early luteal phase (80%) and on production of IGFBP-1 in corpora lutea from the late luteal phase (50%) (P < 0.05), whereas production of IGF-II and IGFBP-3 was not affected by nitric oxide. In conclusion, the components of the IGF-IGFBP system are expressed in the human corpus luteum throughout its lifespan. Nitric oxide regulates IGF-I and IGFBP-1 production, indicating that the growth factors may serve, at least in part, as mediators of the action of nitric oxide in the human corpus luteum.     

Spatial and temporal expression of insulin-like growth factor-I, insulin-like growth factor-II and the insulin-like growth factor-I receptor in the sheep fetal mammary gland

The mammary gland is an example of a tissue of epidermal origin that depends for the development of its characteristic morphology on underlying mesenchymal cells. The interaction between mesenchyme and epithelium appears to be mediated by polypeptide growth factors. In situ hybridization has been used to study, in the mammary gland of female sheep fetuses, the distribution of mRNA for the mammary mitogens, insulin-like growth factor (IGF)-I and IGF-II, and the IGF-I receptor, from 10 to 20 weeks of intrauterine life (term is approximately 22 weeks). At 10 weeks, secondary ducts had formed from the primary duct. By week 20, the gland had increased in volume and complexity, showing primitive lobules embedded in intralobular connective tissue disposed around main ducts. IGF-I and IGF-II mRNA were expressed in cells of the intralobular connective tissue underlying the epithelium, while the IGF-I receptor was expressed in epithelium. Quantitation by absorbance measurements showed that mRNA expression increased with pregnancy stage for IGF-I and IGF-II, but not significantly for the IGF-I receptor, and that IGF-II was more highly expressed than IGF-I. A role for the IGF system in mediating mesenchymal epithelial interactions in mammary development is indicated.     

Insulin-like growth factors-I and -II and insulin-like growth factor-binding protein-2 in dominant equine follicles during spring transition and the ovulatory season

The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.     

Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d −20, −10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply correspond in the brain. We thus assume independent control of IGFBP distribution between plasma and CSF. Due to the known anorexic effect of IGF-I, elevated plasma concentrations of IGFBP-2 and IGFBP-4 during the postpartum period in conjunction with reduced plasma IGF-I concentrations may be interpreted as an endocrine response against negative energy balance in early lactation in dairy cows.     

Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles

Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.     

Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at oestrus and during early pregnancy

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.     

Influence of ad libitum milk replacer feeding and butyrate supplementation on the systemic and hepatic insulin-like growth factor I and its binding proteins in Holstein calves

Ad libitum milk feeding and butyrate (B) supplementation have the potential to stimulate postnatal growth and development in calves. The somatotropic axis is the main endocrine regulator of postnatal growth and may be affected by both ad libitum milk replacer (MR) feeding and B supplementation in calves. We hypothesized that ad libitum MR feeding and B supplementation stimulate systemic and hepatic insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP) in preweaning calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (6 L/d; Res). In each feeding group half of the calves received a MR with 0.24% butyrate and the other half received same MR without butyrate. Ad libitum MR feeding was performed from d 4 until wk 8 of age. From wk 9 to 10, Adl and Res calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Feed intake was measured daily and body weight weekly. Blood samples for analyzing plasma concentrations of glucose, insulin, IGF-I, and IGFBP-2, -3, and -4 were taken on d 1, 2, 4, and 7, then weekly or every other week (IGFBP) until wk 11 of life. Liver samples were taken on d 50 and at the end of the study (d 80) to measure gene expression of the growth hormone receptor 1A (GHR1A), IGF1, IGFBP1 to 4, and of the IGF Type 1 and insulin receptor in the liver. Intake of MR and body weight were greater, but concentrate intake was lower in Adl than in Res. Plasma concentrations of IGF-I and IGFBP-3 were greater and plasma concentration of IGFBP-2 was lower in Adl than in Res during the ad libitum milk feeding period. After reduction of MR in both groups to 2 L/d plasma concentrations of IGF-I and IGFBP-4 were lower and plasma concentration of IGFBP-2 was higher in Adl than in Res. Supplementation of B depressed plasma IGF-I from wk 1 to 4 and in wk 9. On d 50, mRNA abundance of the GHR1A and IGF1 was greater and of IGFBP2 mRNA was lower in Adl than in Res. At d 80, IGFBP2 mRNA was greater in Adl than in Res, and IGFBP2 mRNA increased with B supplementation. Ad libitum MR feeding stimulated the systemic and hepatic IGF system and mirrored the greater growth rate during the ad libitum MR feeding, whereas butyrate supplementation partly reduced the systemic and hepatic IGF system.     

Growth hormone,insulin-like growth factor I and cell proliferation in the mouse blastocyst

The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alpha IR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alpha IR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, GH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.     

Steroidogenic responses of pig corpora lutea to insulin-like growth factor I (IGF-I) throughout the oestrous cycle

This study was designed to investigate the roles of insulin-like growth factor I (IGF-I), IGF-type I receptor (IGF-IR) and IGF-binding proteins (IGFBPs) in regulating progesterone secretion by pig corpora lutea during the oestrous cycle, and the signal transduction pathways involved in mediating the steroidogenic actions of IGF-I. Corpora lutea were collected on days 4, 7, 10, 13 and 15 or 16 of the oestrous cycle, enzyme dissociated and the luteal cells were cultured for 24 h in Medium 199 with IGF-I (0-100 ng ml(-1)), long R(3)-IGF-I (0-100 ng ml(-1)), anti-IGF-I (Sm 1.2B; 0-10 microg ml(-1)), anti-IGF-IR (alphaIR3; 0-2 microg ml(-1)), or IGF-I signal transduction pathway inhibitors (phosphatidylinositol (PI)-3-kinase: 100 nmol Wortmannin l(-1) and 10 micromol LY 294002 l(-1); MAP kinase: 50 micromol PD 98059 l(-1)) to investigate their effects on IGF-I (100 ng ml(-1)) stimulated progesterone secretion. Pig luteal cells displayed dose-dependent responses to IGF-I and long R(3)-IGF-I on days 4 and 7 of the oestrous cycle, but not on days 10-16. There was no difference in the ED(50) or V(max) (maximal response) values between IGF-I and long R(3)-IGF-I. Neither anti-IGF-I nor anti-IGF-IR had significant effects on progesterone secretion, at any dose or day. Wortmannin and LY 294002 blocked IGF-I stimulated progesterone secretion, but PD 98059 was without effect. Finally, IGF-I (6 microg) infused into the ovary on day 7 in vivo significantly increased progesterone secretion within 45 min of infusion. The conclusions of this study are: (1) IGF-I has steroidogenic actions only on 'young' (days 4-7) pig corpora lutea; (2) endogenous IGF-I and IGFBP are insufficient to modulate progesterone secretion in vitro; and (3) the steroidogenic actions of IGF-I are mediated via PI-3-kinase.     

Endocrine disruption of uterine insulin-like growth factor expression in the pregnant gilt

Early exposure of pregnant gilts to oestrogen, prior to the normal period of porcine conceptus oestrogen secretion, disrupts the uterine environment resulting in complete embryonic mortality during the period of placental attachment to the uterine surface. The current study evaluates the uterine insulin-like growth factor (IGF) system following endocrine disruption of early pregnancy in gilts through exposure to exogenous oestrogen on Days 9 and 10 of gestation. Endometrial IGF gene and protein expression, IGF-I receptor (IGF-IR) gene expression, and uterine lumenal content of IGF binding proteins (IGFBPs) were evaluated in control and oestrogen-treated gilts on Days 10, 12, 13, 15 and 17 of gestation. Oestrogen treatment altered endometrial IGF-I and IGF-IR gene expression on Days 12 and 13 of gestation. Uterine content of IGF-I and IGF-II in control gilts was greatest on Days 10, 12, and 13 followed by a four- to sixfold decrease on Day 15 of gestation. Oestrogen treatment caused a premature proteolysis of IGFBPs within the pregnant pig uterus on Day 10 of gestation, and an earlier decline in uterine lumenal IGF-I content. Results demonstrate that early exposure of pregnant gilts to oestrogen causes premature loss of uterine IGFs during the period of conceptus elongation. Timing for the release of uterine IGFs during early porcine conceptus development may play an important function in the ability of the conceptus to attach and survive during the establishment of pregnancy.     

Autocrine insulin-like growth factor II inhibits beta-casein mRNA expression in a mammary cell line

A hallmark of mammary cell differentiation is the induction of beta-casein mRNA expression. A mouse mammary epithelial cell line (COMMA-1D) was treated with insulin, hydrocortisone (HC), and prolactin (Prl) at concentrations (50, 500, and 20 ng/ml, respectively) that resulted in less than half-maximal beta-casein mRNA expression. The cells secreted insulin-like growth factor (IGF)-II (106 pg/ml per 24 h) in the condition media under these conditions. Replacement of insulin with rhIGF-II (150 ng/ml) resulted in significantly less beta-casein mRNA expression. Long-Arg IGF-I (50 ng/ml) was similar to insulin in terms of its ability to induce differentiation, but its activity differed from that of insulin in that it also induced cell proliferation. When the two receptor-specific IGF-II analogs, Arg54,55IGF-II and Leu27IGF-II, were used in studies, only at high concentrations (150 ng/ml) was either analog capable of stimulating any beta-casein mRNA expression. When autocrine IGF-II was immuno-neutralized or bound by the addition of rhIGF binding protein-3 (IGFBP-3)beta-casein mRNA expression was enhanced seven-fold and three-fold, respectively. Exogenous application of IGF-II to counteract the IGF-II mAb stimulation resulted in increased cellular growth and reduced differentiation. We conclude that autocrine IGF-II inhibits mammary cell differentiation and that the blockage of autocrine IGF-II benefits mammary cell differentiation.     

A homologous radioimmunoassay for quantification of insulin-like growth factor-binding protein-2 in blood from cattle

Insulin-like growth factor-I and -II (IGF-I, IGF-II) circulate in biological fluids bound to six different IGF-binding proteins that regulate IGF bioactivity. The IGF-binding protein-2 is regulated by growth hormones, and its concentration depends on nutrition and physiological state. Specific antibodies directed against bovine IGF-binding protein-2 were produced, and IGF-binding protein-2 levels in bovine blood samples were quantified by radioimmunoassay. Parallel displacement curves showed strong cross-reactivity with bovine and ovine plasma, were low with porcine plasma, and no cross-reactivity with rat or chicken plasma. Addition of IGF-I or -II to a control pool of bovine plasma did not significantly alter control IGF-binding protein-2 values in a radioimmunoassay. Six nycthemeral periods, determined for three young bulls bled on two occasions, showed that IGF-binding protein-2 plasma levels were stable throughout the day; two or three samples were sufficient to characterize the animal. Cows treated with recombinant bovine somatotropin (bST) had significantly lower serum levels of IGF-binding protein-2 than did control cows. Furthermore, IGF-binding protein-2 levels were dramatically increased at the onset of lactation. This radioimmunoassay for bovine IGF-binding protein-2, which enables quantitative assessment of IGF-binding protein-2 concentration in cattle, confirmed that IGF-binding protein-2 concentrations are depressed by administration of bST, enhanced after calving, and showed absence of diurnal variation.     

Expression of growth hormone receptor 1A messenger ribonucleic acid in liver of dairy cows during lactation and after administration of recombinant bovine somatotropin.

The mRNA for growth hormone receptor is transcribed from at least three different promoters in cattle. The first promoter (P1) is liver-specific and transcribes growth hormone receptor mRNA containing exon 1A (growth hormone receptor 1A). The second and third promoters (P2 and P3) are active in a variety of tissues and transcribe growth hormone receptor mRNA containing exon 1B and 1C. The objective was to characterize P1 activity by measuring the amount of growth hormone receptor 1A mRNA in liver of dairy cows at different stages of lactation as well as after administration of recombinant bovine somatotropin (rbST). In study 1, liver RNA was isolated from Holstein cows during the dry period (nonlactating, n = 6) and during early (n = 6), mid (n = 6), and late (n = 11) stages of lactation. Six of the late-lactation cows received injections of rbST (25 mg/d) for 7 d prior to collection of liver tissue. In study 2, lactating Holstein cows received either no infusion (control, n = 10) or continuous infusion of rbST (29 mg/d, n = 10) for 63 d. The amount of growth hormone receptor 1A mRNA was decreased in early- and mid-lactation cows compared with late-lactation cows or nonlactating cows (study 1). Administration of rbST increased growth hormone receptor 1A mRNA (studies 1 and 2). The total amount of growth hormone receptor transcribed from alternative promoters (growth hormone receptor P2 and P3) remained unchanged during different stages of lactation or in response to rbST. We conclude that changes in liver growth hormone receptor mRNA in lactating dairy cattle primarily depend on growth hormone receptor P1 activity.