Receptors In Mammary Gland Research Articles

Effect of chronic thyroxine treatment on IGF-I, IGF-II and IGF-binding protein expression in mammary gland and liver during pregnancy and early lactation in rats

Hyperthyroidism in rats produces organ hypertrophy and increases in circulating IGF-I and IGF-binding protein (IGFBP)-3. Chronic treatment with thyroxine (T(4)) during pregnancy advances parturition, blocks lactation and changes several hormone receptors in mammary gland and liver. Since IGFs are implicated in mammary and liver growth and in differentiation, we studied the effects of hyperthyroidism, induced by daily injections of T(4) (0.25 mg/kg). Using quantitative RT-PCR and in situ hybridization, the gene expression of IGF-I, IGF-II and the IGFBPs was determined in mammary gland and liver of rats at estrus and days 7, 14 and 21 of pregnancy (G7, G14, G21), day 1 postpartum (L1) and 3 days after removing the litter (L4). Circulating levels of IGF-I, tri-iodothyronine (T(3)), PRL and GH were measured. T(4) treatment (HT) increased circulating T(3) save on G21, did not change serum IGF-I, increased PRL on G21 and decreased GH on L1. PRL decreased on L1 because of the absence of lactation. Hepatic IGF-I mRNA was low during pregnancy and increased on L4. HT advanced this increase to L1. In controls, liver IGFBP-3 mRNA levels decreased from G14 to G21, whereas IGFBP-4 showed an inverse pattern. HT lowered IGFBP-3 mRNA and increased IGFBP-4. Increases in mammary concentrations of IGF-I, IGFBP-3 and IGFBP-4 mRNAs were seen on G21. HT delayed these peaks to L1. Mammary IGF-II and IGFBP-2 mRNA levels were high on G7 and G14, and fell afterwards, with HT having no effects. IGFBP-5 mRNA decreased during pregnancy and increased on L1. HT increased IGFBP-5 levels in early pregnancy and on L1. IGF-I mRNA localized to connective and epithelial mammary tissue, while IGFBP-2 and IGFBP-5 mRNA was only in epithelial cells. These results imply a role for IGF-I, IGFBP-3 and IGFBP-4 in terminal mammary development, while IGF-II and IGFBP-2 may be implicated in early growth. IGFBP-5 has been implicated in mammary apoptosis, and the HT-induced increase may play a role in the premature mammary involution of the HT rats.

Progesterone receptors in normal, dysplastic and tumourous feline mammary glands. Comparison with oestrogen receptors status

The aims of the study were to standardise an immunohistochemical (ihc) method for the detection of progesterone receptors (pr) on formalin-fixed, paraffin wax-embedded tissue sections of feline mammary gland tumours and dysplasias, comparing the results with those obtained using the radiolabelled ligand dextran coated charcoal (dcc) assay applied to frozen tissue samples from the same cases. Also, to define the immunohistochemical distribution of pr in the different cellular compartments of the lesions and to compare the oestrogen receptor (er) and pr status of the feline mammary lesions. Proliferative mammary lesions collected from 34 cats were studied; 25 malignant tumours and 9 benign tumours and dysplasias. pr protein was present at a concentration of 5fmolmg−1 (positivity threshold) in 37·5 per cent of malignant tumours and 66·7 per cent of benign tumours and dysplasias while immunoreative products to pr antibody were found in the nuclei of tumour cells in 38·5 per cent and 66·7 per cent of the cases, respectively. Concordance between dcc-pr and ihc-pr was 88·5 per cent (P<0·001). The specificity (true negatives) and sensitivity (true positives) of the ihc method were 89·4 per cent and 87·5 per cent respectively. The presence of pr was linked to the absence of ovariectomy (P<0·02). Oestrogen receptors, detected by either method, were also detected in half the cases in which pr had been detected. In malignant tumours, the most prevalent groups were the er + pr + ander −pr + groups.

Cellular expression of estrogen and progesterone receptors in mammary glands: regulation by hormones, development and aging

At present, there is an extensive body of literature documenting the participation of estrogen receptors (ER) and progesterone receptors (PR) in mammary gene expression. Yet, the precise roles of these receptors in regulating mammary development, carcinogenesis and the growth of a subset of tumors still remain unclear. Mammary glands are composed of various cell types with different developmental potentials. Further, ultimately, that it is their mutual interactions which dictate the behavior of mammary epithelial cells. Therefore, to resolve the roles of ER and PR in normal mammary growth, differentiation and carcinogenesis, analyses for the expression of these receptors at the level of individual cell types is of paramount importance. Accordingly, in the present studies using immunolocalization techniques, we document the ontogeny and cellular distribution of ER and PR during mammary development and in response to ovarian hormones and aging. In addition, we discuss the potential biological significances of the expression patterns of ER and PR during various physiological states. We believe that the observations reported here should provide a conceptual framework(s) for elucidating the roles of ER and PR in normal and neoplastic mammary tissues.

Effect of chronic thyroxine treatment on pregnancy in rats: effects on oestrogen, progesterone, prolactin and gh receptors in uterus, liver and mammary gland

We have previously shown that experimental hyperthyroidism produces premature and difficult delivery and absence of lactation in spite of apparently adequate luteolysis and lactogenesis. To study the possible causes of these alterations we measured the effect of treatment with T4 (0.25 or 1 mg kg−1, s.c., daily, started 10-15 days before mating, HT0.25 and HT1) on serum hormones and their receptor (R) concentrations in reproductive tissues on day 20 of pregnancy (1800 hours), comparing them with controls on the same day (C20), or on day 21 of pregnancy (1800 hours) (C21). Serum prolactin (PRL) and corticosterone (B) concentrations increased in the HT groups, progesterone (Pg) and GH decreased and estradiol (E2) did not change, compared with C20 group. C21 rats had increased serum PRL and decreased Pg and GH. In HT rats mammary DNA and protein tissue content was doubled. Receptor concentrations were expressed per mg DNA. Mammary PRL-R were increased in HTI rats, while E-R and Pg-R were significantly lower in both HT groups. HT0.25 and HT1 rats had increased uterine E-R and Pg-R and decreased liver PRL-R and GH-R as well as their mRNAs. Liver E-R, PRL-R and GH-R were decreased in C21 rats, while uterine Pg-R were increased. Thus, some of the observed changes (serum Pg and GH, mammary and uterine Pg-R, and liver GH-R and PRL-R decreases and serum PRL increase) may be due at least partially to the advancement in luteolysis and delivery, being similar to the changes observed between days 20 and 21. The changes in serum B, mammary PRL-R, and mammary and uterine E-R may be caused solely by the T4 treatments and may play a role in the alterations previously observed.

In situ localization of progesterone receptors in normal mouse mammary glands: Absence of receptors in the connective and adipose stroma and a heterogeneous distribution in the epithelium

In normal mammary glands of both rodents and humans, progesterone promotes the proliferation of epithelial cells and several lines of evidence suggest that this action of progesterone may be mediated by progesterone receptor (PR). It is well established that normal mammary development involves a complex interplay between the epithelial cells and the surrounding fatty stroma. Furthermore, during mammary development, there is a change in both the relative proportion of epithelial cells and the steady-state levels of PR. Therefore, towards understanding the precise role of PR in mammary development, we have generated a highly sensitive antibody against mouse PR and examined its pattern of localization. Immunoreactive PR was detected only in the epithelial cells of the ducts while both the adipose and fibrous stroma surrounding these ducts were receptor negative. Similarly, PR mRNA was also associated only with the ductal epithelial cells. Approximately only 45–50% of the ductal cells were receptor positive and this distribution remained unchanged whether or not the tissues had been exposed to estrogen, suggesting that they may represent a distinct subpopulation. The potential significance of these findings to mammary development is discussed.

S24-5 - Interaction between myoepithelial and secretory epithelial cells via purinergic receptors in mammary gland.

S24-5 - Interaction between myoepithelial and secretory epithelial cells via purinergic receptors in mammary gland.

Histamine receptors in human mammary gland, different benign lesions and mammary carcinomas

Histamine receptors in human mammary gland, different benign lesions and mammary carcinomas

The Rabbit Mammary Gland Prolactin Receptor Is Tyrosine-phosphorylated in Response to Prolactin in Vivo and in Vitro

We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.

Detection of Messenger RNA for Gonadotropin-Releasing Hormone (GnRH) but Not for GnRH Receptors in Mouse Mammary Glands

While gonadotropin-releasing hormone (GnRH), GnRH-like, or GnRH receptor (GnRH-R) have been reported to exist in several tissues other than brain or anterior pituitary, there is no report concerning GnRH or GnRH-R gene expression in the normal mammary gland. In order to define the production of GnRH as well as GnRH-R in the mammary gland at the molecular level we examined their gene expression in various functional stages of the mouse mammary gland using the reverse transcriptase-polymerase chain reaction (RT-PCR). GnRH mRNA transcripts were found in mouse mammary glands of mid-pregnant, lactating, and 3, 6, 9 days post-lactational mice, whereas GnRH-R mRNA transcripts were not detected in mammary glands of any functional stage. These results suggest a possible biological role of GnRH in mammary gland.

Growth factors and their receptors in neoplastic mammary glands

Control of the growth of mammary glands is largely exerted in vivo by systemic hormones and locally-produced growth factors, whereas malignant tumours gradually lose the ability to respond to both types of control in vivo. However, the systemic hormones have little direct effect on stimulating the growth of rat or human mammary cell lines in vitro. Estrogens are thought to work by stimulating locally-produced growth factors and/or their receptors, eg transferrin, TGF alpha and IGF-1, and prolactin by a contaminating pituitary mammary growth factor (PMGF). Mammary stem cells intermediate between epithelial and myoepithelial cells are thought to be retained in malignant carcinomas, whereas the TGF alpha and bFGF-producing myoepithelial cells are lost. Hormonal autonomy of carcinomas may develop by overproduction of the locally-produced growth factors, their receptors (including related receptors, eg c-erbB-2) and/or by stem cells differentiating sufficiently to utilise normal control mechanisms, eg refractivity to PMGF and autocrine/paracrine response to bFGF. The failure of the stem cells to differentiate completely to myoepithelial cells in carcinomas greatly reduces the heparan sulphate proteoglycan sink used to sequester to bFGF in normal glands and also removes the possibility of eliminating cells by terminal differentiation, both processes possibly contributing to the uncontrolled growth of the malignant breast cell.

Mechanism of Prolactin Action on the Dissociation of Prolactin from Rabbit Mammary Gland Receptor

In order to elucidate the action of prolactin (PRL) on the dissociation of PRL from the receptor, Triton-solubilized and Concanvalin A-purified rabbit mammary gland PRL receptor was immobilized onto a nitrocellulose membrane, and the dissociation of PRL was determined in the presence of PRL. PRL accelerated the dissociation of PRL in a concentration dependent manner for the first 3 h of incubation. The effects of PRL on dissociation were independent of the occupancy of PRL at the zero-time dissociation. In particular, PRL-dependency was influenced by the PRL-binding capacity of the membrane. The dissociation reaction was PRL-independent in the low-binding capacity membrane, indicating that PRL itself had no accelerating actions. With greater binding capacity, the dissociation reaction was seen to become PRL-dependent. It was concluded that PRL accelerated the dissociation of PRL through the formation of PRL-receptor complex when the receptor was located closely to the neighboring receptors.

1,25-Dihydroxyvitamin D radioreceptor assay using bovine mammary gland receptor and non-high performance liquid chromatographie purification

1,25-Dihydroxyvitamin D radioreceptor assay using bovine mammary gland receptor and non-high performance liquid chromatographie purification

Influence of ovariectomy on the effect of bovine growth hormone (bGH) treatment on mammary growth, serum hormones, and liver and mammary gland receptors in prepubertal Holstein heifers : S. Purup 1, K. Sejrsen 1 and R.M. Akers 2. 1National Institute of Animal Science, 8830 Tjele, Denmark and 2Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA

Influence of ovariectomy on the effect of bovine growth hormone (bGH) treatment on mammary growth, serum hormones, and liver and mammary gland receptors in prepubertal Holstein heifers : S. Purup 1, K. Sejrsen 1 and R.M. Akers 2. 1National Institute of Animal Science, 8830 Tjele, Denmark and 2Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA

The ontogeny and cellular distribution of estrogen receptors in normal mouse mammary gland

The appearance, epithelial and stromal cell distribution of estrogen receptors (ER) in normal mouse mammary gland were determined between 1 and 10 weeks of age using immunohistochemistry. The effect of ovariectomy and estrogen (E)-treatment on the distribution and concentration of ER-positive cells at various ages was also analyzed. These studies demonstrate that ER are present in both mammary epithelial and stromal cells before the mammary gland exhibits a proliferative response or increase in progesterone receptor concentration as a result of E-treatment. Furthermore, an analysis of E-treatment suggests that although ER are present at an early age, there may be additional factors that determine the nature and extent of E-responsiveness.

Pregnancy-dependent initiation in tumorigenesis of Wistar rat mammary glands by 60Co-irradiation

Pregnant Wistar rats received whole body irradiation with 260 cGy gamma-rays at days 7, 14 and 20 of pregnancy and then were treated with diethylstilbestrol (DES) for 1 year. The highest incidence (92.9%) for tumorigenesis of mammary glands was observed in the rats irradiated in late pregnancy. Histological examination showed that tumors were classified as fibroadenoma and adenocarcinoma. To determine the reasons for specific induction of mammary tumors by irradiation in late pregnancy, hormone concentrations in serum and estrogen receptors in mammary glands during pregnancy were measured. Concentrations of estradiol, progesterone, 11-deoxycorticosterone and placental lactogen at day 20 were higher than at days 7 and/or 14, but no difference was observed in the concentrations of prolactin and thyroid-stimulating hormone during pregnancy. The estrogen receptor in mammary glands at day 20 was indicated to have the highest affinity and the highest binding capacity during pregnancy. Normal mammary glands at day 20 were suggested to have more abundant epithelial cells in the mammary lobes than those at days 7 and 14. The data suggest that the critical requirements for the initiation of tumorigenesis by gamma-rays are dependent upon the differentiated state of mammary glands exposed to various hormones, and that the concentration and persistence of the synthetic estrogen (DES) are necessary for the promotion of tumorigenesis of the irradiated mammary glands.

Comparison of binding affinities of a series of oxytocin analogues to uterine and mammary gland receptors and their biological potencies

The affinity of 12 oxytocin analogues of similar structure but differences in conformational freedom and agonistic and antagonistic properties to receptors in guinea pig uterus and rat mammary gland membrane preparations was determined by competitive binding experiments using tritiated oxytocin. The values obtained for the apparent dissociation constant KD were compared to the values of biological activities from classical pharmacological tests ( uterotonic test in vitro and galactogogic test in vivo).

Characterization and applications of monoclonal antibodies to the prolactin receptor.

Monoclonal antibodies (mAbs) were produced in BALB/c mice immunized with partially purified PRL receptors from rat liver. Two mAbs (T1 and T6) were able to completely inhibit [125I]ovine PRL ([125I]oPRL) binding to solubilized rat liver PRL receptors, while two other mAbs (U5 and U6) showed only a small effect on PRL binding, but were able to precipitate hormone-receptor complexes. Scatchard analysis of [125I]oPRL binding to rat liver microsomes in the presence of mAbs resulted in a decrease in the number of sites without changing the affinity of PRL binding by T1 and T6, whereas U5 and U6 altered neither parameter. [125I]mAb binding to rat liver microsomes was performed in the presence of various concentrations of unlabeled mAbs or oPRL to examine the interaction between mAbs. Competition of binding to the receptor was observed, respectively, between T1 and T6, U5 and U6, and U5 and E21 (a mAb to the rat liver PRL receptor previously produced). Both [125I]T1 and [125I]T6 binding were inhibited by oPRL, although not completely (80% inhibition at the higher concentrations). When [125I]T1 binding was analyzed by Scatchard analysis, two classes of binding sites to rat liver microsomes were found, of which only the number of higher affinity sites was affected by the presence of oPRL in incubation. Similar results were observed for [125I]T6 binding. [125I]mAb binding to microsomes from other tissues and species was examined. All five mAbs were able to bind to microsomes from rat tissues (liver, ovary, adrenal, prostate, and Nb2 lymphoma cells), similar to the level of [125I]oPRL binding in these tissues. The binding characteristics of [125I]T6 or [125I]U5 were essentially identical in all rat tissues examined. Although T1, U6, and E21 showed strong species specificity, there was significant binding of T6 to rabbit liver and mammary gland and of U5 to rabbit and pig mammary gland and mouse liver Competition curves of [125I]U5 binding were parallel for rat, rabbit, and mouse tissues, while [125I]T6 binding was able to distinguish PRL receptors in rabbit mammary gland from those in rat tissues. The use of 125I-labeled mAb in immunoblot analysis of the PRL receptor resulted in a marked increase in sensitivity. All mAbs detected microsomal PRL receptors in rat liver with mol wt of 84,000, 42,000 and 40,000. As little as 4 fmol receptors can be identified using this approach. Microsomal PRL receptors from rat ovary, prostate, and Nb2 cells and purified receptors from pig and rabbit mammary gland were subjected to immunoblot analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland.: Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)51[Tyr(Me)2, Thr4,Tyr-NH29]OVT

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone effects on deoxyribonucleic acid synthesis in normal mouse mammary glands.

Current in vivo and in vitro data indicate that progesterone (P) is required for epithelial proliferation in mammary glands of adult mice. P receptors (PgR) in adult mammary glands are under estrogenic regulation. By contrast in immature, pubertal 5-week-old mice, PgR are present only in low concentration and cannot be increased by estrogen (E). Having identified two different mammary gland developmental states with differing PgR compositions, the purpose of the present studies was to investigate the roles of E-dependent and -independent PgR in epithelial cell proliferation. DNA histoautoradiography was used to quantitate the effect of P on DNA synthesis; hormonal effects on mammary gland morphology were also assessed. The results demonstrate the P, rather than E, has a major role in stimulating epithelial DNA synthesis in the adult mammary gland. The morphological correlate of P's stimulatory effect on cell proliferation was increased ductal side-branching. We postulate that P is acting via E-dependent PgR and that one important way in which E promotes epithelial cell proliferation in adult tissue is by its ability to increase PgR concentration. In the immature mammary gland E rather than P is primarily responsible for stimulation of epithelial cell proliferation at this developmental stage. It appears likely that the absence of a predominant, P-mediated cell proliferative effect is related to the lack of E-dependent PgR.

Evolution of lactogen receptors in selected rabbit tissues during pregnancy.

The ontogeny of lactogen receptors in brain, adipose, liver, kidney, adrenal gland, mammary gland, ovarian and uterine tissues of pregnant rabbits was evaluated in this study using 125I bovine prolactin as tracer. Brain and adipose tissues were found to have very low receptor numbers throughout pregnancy (less than 20 fmol/mg of protein), while liver and kidney had higher but constant levels of receptor through the same period (200 and 100 fmol/mg of protein, respectively). Mammary gland and adrenal gland tissues exhibited sharp increases in prolactin binding between 15 and 17 days with both having peak receptor binding at 17 days of around 200 fmol/mg of protein. Ovarian and uterine receptor binding increased slowly after day five of pregnancy and reached peak levels of approximately 225 fmol/mg of protein at day 20. Scatchard analysis of the binding of protein in the tissues having increased binding during the course of pregnancy, revealed that its affinity for sites in these tissues was the same at 5 and 20 days of gestation, indicating the rise in binding to be a result of increased numbers of available receptors. Sub-organ localization studies found the binding of prolactin to adrenal gland, ovary and uterus to be essentially located in adrenal cortex, nonluteal ovary and endometrium. Incubation of membranes from each of the tissues showing significant change during pregnancy, from several time points of pregnancy, with 5.0 M MgCl2 produced little change in apparent receptor numbers; suggesting that receptor occupancy levels of endogenous prolactin was low.