Effects Of Lactogenic Hormones Research Articles

Effects of tripeptides and lactogenic hormones on oligopeptide transporter 2 in bovine mammary gland

This study was conducted to investigate the expression of oligopeptide transporter 2 (PepT2) and its potential function in bovine mammary gland. First, the PepT2 mRNA and protein were determined in cultured mammary epithelial cells. Then the effects of lactogenic hormones (prolactin, hydrocortisone or insulin) and substrate (threonyl-phenylalanyl-phenylalanine) on PepT2 were investigated. The PepT2 mRNA and protein were successfully detected in bovine mammary epithelial cells. PepT2 gene expression was enhanced by the addition of 50, 500 and 5000 ng/ml prolactin, 10 and 100 ng/ml hydrocortisone, and 50, 500, 5000 and 50,000 ng/ml insulin. PepT2 mRNA abundance was increased when 5, 10 and 15% of threonyl-phenylalanyl-phenylalanine was included. Responses of PepT2 to lactogenic hormones and oligopeptide inferred that it may play an important role in bovine mammary gland.

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte-derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry-off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.

Gene expression during involution of mammary gland (review).

After cessation of lactation, the mammary gland undergoes involution, regressing to a state resembling that of a virgin animal. This phase of mammary gland development is characterized by epithelial cell death and tissue remodeling. To understand molecular mechanisms of mammary gland involution, we identified involution-induced clones by differential screening of a mouse mammary gland cDNA library. Several known genes were induced during mammary gland involution: sulfated glycoprotein-2 (SGP-2), WDNM1, lactoferrin, ferritin heavy chain (FHC), lysozyme and osteopontin genes. Involution of the mammary gland is presumed to be mediated by a decrease in serum prolactin level induced by weaning, but may also involve changes in paracrine or autocrine growth factors. Effects of lactogenic hormones and EGF on the expression of the involution-induced genes were examined in mammary epithelial cells. Insulin, dexamethasone, and prolactin did not influence the expression of the FHC, WDNM1 and SGP-2 genes. However, EGF strongly inhibited the expression of WDNM1 and SGP-2 genes. Our recent results are reviewed and discussed.

Pretreatment with glucocorticoid is essential for lactogenic induction of the bovine β-casein/cat expression in HC11 cells

Hormonal regulation of the bovine β-casein gene expression was studied in a murine mammary epithelial HC11 cells and compared with that of the rat β-casein gene expression. CAT expression vectors driven by their promoter sequences were transfected into HC11 cells. Stable transfectents were treated with lactogenic hormones, dexamethasone and prolactin for 2 days in confluent cultures. While the lactogenic hormones synergistically induced a strong activation of the rat β-casein/CAT expression, neither a single or combined treatment of dexamethasone and prolactin induced the bovine β-casein/CAT expression. To test a sequential treatment effect of lactogenic hormones on the bovine β-casein/CAT expression, cells were first treated with either dexamethasone or prolactin for various days and then subjected to the second treatment with both hormones for 2 days. Only dexamethasone-, but not prolactin-pretreated cells showed a strong lactogenic induction. Moreover, the fold induction of dexamethasone-pretreated cells increased gradually as a function of duration of dexamethasone pretreatment. A series of the bovine β-casein/CAT constructs with different length of the bovine β-casein 5' flanking region ranged from 0.3 kb to about 15 kb was analyzed in 12-days dexamethasone-pretreated cultures. CAT expression was increased even in 0.3 kb-containing construct, but prominent induction was seen in more than 1.8 kb-containing constructs. Therefore, it could be concluded that a long-term dexamethasone pretreatment is essential for lactogenic induction of the bovine β-casein expression and the 0.3 kb proximal promoter region is important, but more distal promoter elements) is necessary for mediating the coordinated action of lactogenic hormones to the bovine β-casein expression.

Number and size of islets of Langerhans in pregnant, human growth hormone-expressing transgenic, and pituitary dwarf mice: effect of lactogenic hormones

Number and size of islets of Langerhans in pregnant, human growth hormone-expressing transgenic, and pituitary dwarf mice: effect of lactogenic hormones

Hormonal and extracellular matrix regulation of plasminogen activator in a bovine mammary epithelial cell line

The effects of lactogenic hormones on urokinase plasminogen activator (u-PA) produced by bovine mammary epithelial cells (MAC-T) were examined. High levels of u-PA activity were detected in growth arrested cells cultured on plastic. This suggests that high levels of PA activity alone are not sufficient to induce proliferation of bovine mammary epithelial cells. Cells were cultured on various extracellular matrices: plastic, fibronectin, collagen, matrigel, and laminin. Basal levels of u-PA activity in media from MAC-T cells cultured on matrigel were 1.6-, 2-, 2- and 3.5-fold higher than that of cells cultured on plastic, fibronectin, collagen, and laminin, respectively. Insulin increased (P<0.01) mammary epithelial u-PA activity on a per cell basis, an effect observed irrespectively of the type of extracellular matrix onto which cells were cultured. Not unexpectedly, insulin-like growth factor (IGF)-I, Des(1-3) IGF-I and IGF-II increased mammary epithelial u-PA activity on a per cell basis and u-PA mRNA levels, thus, mimicking the effect of insulin. Dexamethasone suppressed (P<0.01) u-PA activity but was unable to suppress the insulin-induced increase in u-PA activity of cells cultured on various extracellular matrices. These data indicate that u-PA activity is modulated by both lactogenic hormones and the type of extracellular matrix.

Regulation of the sheep beta-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-gamma activation site-related element.

Polypeptide and steroid hormones regulate the transcription of milk protein genes in the mammary gland. The promoter sequence motifs and factors through which these hormones mediate their effects in vivo are not clearly defined. Milk protein binding factor (MPBF) is a factor that has recognition sites in the promoters of many milk protein genes including three sites in the promoter of the sheep beta-lactoglobulin (BLG) gene. Mutagenesis of these sites reduced expression of the BLG gene in lactating mammary glands of transgenic mice but did not affect the tissue specificity of the transgene. Furthermore, mutation of all three sites abolished the response of the BLG gene to lactogenic hormones in HC11 mammary cells. Together these results indicate that MPBF mediates the effects of lactogenic hormones in the mammary gland but does not play a role in determining mammary specificity. The similarity between the MPBF binding site and the gamma-interferon activating site suggests that MPBF is related to the STAT family of cytokine-induced transcription factors.

The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages

1. 1. The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ([ 3H]-lysine, [ 14C]-cycloleucine) in rat mammary epithelial cell cultures. The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages. 2. 2. Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake. But increase of these activities was different at each physiological stage. 3. 3. The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation. And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

Identification of two specific prolactin binding sites on Nb2 rat lymphoma cells.

Several laboratories have earlier identified and characterized a specific prolactin binding site (receptor) on Nb2 cells which has a Kd of about 2 x 10(-10) M and a binding capacity of about 12,000 receptor sites per cell. Using [I125]-labeled human growth hormone (hGH), we have confirmed the results of these earlier reports. In addition, a second, "high-affinity" receptor site for prolactin has been identified. This site has a Kd of about 2 x 10(-12) M and there are an average of 67 sites per cell. Binding of [125]-hGH to the high-affinity receptor site is saturated by 1 min after incubation at 37 degrees C, whereas saturation of the lower affinity site requires a 1-3 hr incubation at 37 degrees C. Specificity of prolactin binding to both the high- and low-affinity "receptors" was established by the fact that several nonlactogenic hormones, including bovine growth hormone and insulin, do not displace [I125]-hGH from the receptor sites. When Nb2 cells were incubated for 1 hr at 0 degrees C with 100 pg/ml [I125]-hGH, no binding to the high-affinity receptor was detected, thus demonstrating the temperature dependence of binding to this receptor. The kinetics of binding of hGH to the high-affinity binding site correlates well with the dose-response effects of lactogenic hormones on mitogenesis in the Nb2 cells. In addition, the rapid binding of hGH to the high-affinity receptor may be related to specific effects of lactogenic-hormones which occur within seconds or minutes after adding these hormones to Nb2 cells.

Hydrocortisone induces the synthesis of alpha 2-macroglobulin by rat mammary myoepithelial cells.

The effects of lactogenic hormones on protein secretion by 25.5-G4.2.3 cells, a rat mammary myoepithelial cell line immortalised with a temperature-sensitive T-antigen, were investigated. Insulin, prolactin, estradiol and progesterone had no effect but hydrocortisone induced the secretion of two proteins with molecular masses of 175 kDa (p175) and 146 kDa (p146), 10-30-fold and 5-fold respectively. The induction of p175 and p146 synthesis by hydrocortisone was greater at 39.5 degrees C than at 33 degrees C reflecting the increased differentiation of 25.5-G4.2.3 cells at the higher temperature. Rat mammary epithelial cells did not synthesise p175. After addition of hydrocortisone to 25.5-G4.2.3 cells, there was a lag phase of 10 h before the synthesis of p175 was induced. Half-maximal induction of p175 synthesis required a hydrocortisone concentration of 0.5 microM. p175 was identified as alpha 2-macroglobulin by N-terminal amino-acid sequence determination and immunoprecipitation with a specific antibody. Hydrocortisone induced a 5-kb alpha 2-macroglobulin-specific mRNA transcript in 25.5-G4.2.3 cells. Myoepithelial cells are responsible for synthesising the basement membrane around the rapidly expanding mammary alveoli during pregnancy. Myoepithelial cells also secrete metalloproteinases which are probably involved in turnover of the basement membrane. We suggest that increased levels of hydrocortisone during pregnancy induce the synthesis of alpha 2-macroglobulin, which is believed to be a potent inhibitor of metalloproteinases, by rat mammary myoepithelial cells to reduce proteolytic degradation of the basement membrane.

Effects of Lactogenic Hormones on Morphological Development and Growth of Human Breast Epithelial Cells Cultivated in Collagen Gels

We investigated the hormonal dependence of the growth and morphological development of human breast epithelial cells (HBEC) cultivated in collagen gels floating on serum–free medium. HBEC used had grown out from breast carcinoma tissue and from histologically non–malignant tissue fragments in the same mastectomy specimens. In the presence of ail three lactogenic hormones, hydrocortisone (HC), insulin (INS), and prolactin (PRL), both carcinoma–derived and histologically non–malignant tissue–derived HBEC grew and formed cell masses with protruding tubule–like extensions that consisted of a multilayer of cells. Deletion of any one of the hormones from the medium had no effect on cell growth of HBEC derived from either tissue type. However, morphological development, especially the morphology of the tubule–like extensions, was altered in the histologically non–malignant tissue–derived HBEC by deletion of HC or INS, and by deletion of HC, but not INS, in the carcinoma–derived HBEC. The present results suggest that the carcinomaderived HBEC have decreased responsiveness to INS as compared to that of HBEC derived from histologically non–malignant breast tissue.

Lactogen-induced protein phosphorylation in Nb2 cells

Lactogenic hormones are potent mitogenic agents in the Nb2 rat lymphoma cell line. Because selective phosphorylation/dephosphorylation of specific proteins by many polypeptide hormones appears to be important in regulating cell growth, the effect of lactogenic hormones on protein phosphorylation was studied in Nb2 cells. Human growth hormone (hGH) promoted the phosphorylation of many proteins (2- to 3-fold) and an M r 29,000 species (pp29) (⩾ 10-fold). In growth-arrested cells phosphorylation of pp29 peaked 4 h after addition of hGH whereas that of other phosphoproteins increased steadily. Ovine prolactin, another potent mitogenic hormone, caused a concentration-dependent increase in pp29 phosphorylation with an EC 50 = 10–20 p m. Other agents which are not mitogenic in this cell line, i.e., 12- O-tetradecanoyl-phorbol-13-acetate (200 n m), 8-Br-cAMP (1 m m), and 8-Br-cGMP (1 m m), did not affect pp29 phosphorylation. Dexamethasone (100 n m), which inhibits lactogen-stimulated growth, reduced hGH-stimulated pp29 phosphorylation to control levels. Fractionation of detergent-treated extracts of hGH-treated cells showed that pp29 is associated with the ribosomal fraction. Taken together, these results show that pp29 phosphorylation is selective and is related to cell growth.

Radioautographic identification of lactogen binding sites in rat median eminence using 125I-human growth hormone: evidence for a prolactin "short-loop" feedback site.

The binding characteristics of human growth hormone were exploited to identify radioautographically lactogen binding sites in the rat median eminence. Following systemic injection 125I-human growth hormone bound preferentially to the lateral palisade zone, a region of median eminence rich in dopamine and LHRH. Coinjection of 125I-human growth hormone with an excess of unlabeled human growth hormone or ovine prolactin, but not bovine grown hormone, competitively blocked 125I-human growth hormone binding to the external median eminence. These observation provide direct evidence of recognition sites for lactogenic hormones in a discrete region of the median eminence associated with hypothalamic regulation of hypophyseal prolactin and luteinizing hormone secretion. Median eminence lactogen binding sites may mediate presumed direct effect of lactogenic hormones on the reproductive functions of the hypophysiotropic hypothalamus.

The effect of lactogenic hormone on liver glycogen and blood glucose in ovariectomized mice.

The effect of lactogenic hormone on liver glycogen and blood glucose in ovariectomized mice.

Effect of Lactogenic Hormone, Growth Hormone and Thyroxine in the Lactating Albino Rat.

Effects of lactogenic hormone (LGH) alone and in combination with growth hormone (GH) and L-thyroxine (T4) upon lactation in the rat have been studied. All rats were nursed on day 14 of lactation with aid of 1 USP unit of oxytocin (OXT) under standardized conditions. One mg LGH alone did not show galactopoietic effects when injected daily from day 7–14. Under combined action of 1 mg LGH and 1 mg GH/day from 7–14, litter weight gain and amount of milk on day 14 have been significantly increased. Addition of 3.0 μg T4/100 g body weight/day from day 7–14 to LGH plus GH resulted in a more highly significant increase in milk yield in comparison to oxytocin alone than did treatment with LGH plus GH. The increase in mean milk yield of 84% over untreated controls by 4 hormones is the highest so far attained in laboratory.

Effect of Lactogenic Hormone on Embryonic Tissues Cultivated in vitro.

Since lactogenic hormone is known to produce a specific effect on epithelial cells of the pigeon cropin vivo, experiments were designed to determine whether it would stimulate the growth of embryonic pigeon crop epithelium cultivated in vitro. If stimulation could be observed in this manner such a test might be more delicate and superior to the present methods employed for determining hormonal potency. Preliminary to testing the hormone on the specific crop epithelium, which reacts to it in vivo, experiments were carried out on the following non-specific tissues cultivated in vitro: (1) Embryonic chick connective tissue, (2) embryonic chick epidermis, (3) embryonic pigeon oesophageal epithelium. Finally the hormone was tested on cultures of embryonic epithelium taken from the crop sac areas known to be reactive in vivo in the hatched pigeons. In all cases the dilutions were performed in triplicate. Also, every experiment was repeated 3 times before the results were reported. Each flask contained about 12 fragments of tissue. (1) Embryonic Chick Connective Tissue. Chick heart tissue from 10- to 14-day-old embryos was used as the source of fibroblasts. The tissues were finely cut into fragments of about 1 mm. in diameter and immersed in Tyrode solution until ready for use. The embryonic fluid was prepared by mincing 14-day-old chick embryos in a tissue grinder and suspending the pulp in 4 parts of Tyrode solution. The suspension was centrifugated at high speed to throw down all cellular material after which the clear supernatant fluid was carefully removed with a pipette. This clear liquid was used for coagulating the plasma as well as a nutrient for the tissue cells. Guinea pig plasma was used in all experiments. This was collected by first drawing 1.0 cc. of a 1-1,000 solution of heparin into a 10.0 cc. syringe and then heart blood to the 10.0 cc. mark. The mixture was quickly centrifugated, the clear plasma removed with a pipette and kept in ice water until ready for use.