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To determine expression and localization of receptors for estrogen (ER), progesterone (PR) and androgen (AR), detailed immunohistochemical evaluations were performed in the Sprague-Dawley rat oviduct during pre- and neonatal development, estrous cycle and pre-implantation period. In addition, real-time RT-PCR studies were conducted to evaluate changes in ERalpha, ERbeta, total PR (PR-A+B), PR-B and AR mRNA expressions. All receptors except for ERbeta were detected in epithelial, and stromal or mesenchymal cells of the fetal and neonatal oviduct, and increased with development. During the estrous cycle and early pregnancy, ERalpha and PR-A+B were expressed in epithelial, stromal and muscle cells throughout the oviduct region, and showed changes in expression predominantly in the isthmus. Only a few epithelial cells in the infundibulum (inf) and ampulla (AMP) showed ERbeta staining. AR was detected in stromal and muscle cells throughout the oviduct region, and in epithelial cells of the inf/AMP. Taken together, ERalpha, PR-A+B and AR were detected in the epithelium of the inf/AMP region, but all of these receptors were expressed in a distinct subset of epithelial cells which were negative for beta-tubulin IV, a ciliated epithelial cell marker. These results contribute to a better understanding of the respective roles of ERs, PRs and AR in the rat oviduct.
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ABSTRACT
Two marsupial casein genes have been isolated from a tammar wallaby (Macropus eugenii) mammary gland cDNA library. Comparisons of the tammar α- and β-casein genes with their eutherian homologues reveal extensive divergence at the levels of nucleotide and amino acid sequences. Regions of similarity between the tammar and eutherian Ca2+-sensitive caseins are restricted to the major phosphorylation sites and the signal peptides. Quantification of casein mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the β-casein gene is dependent upon prolactin and insulin, while maximal induction of the α-casein gene is dependent upon prolactin, insulin and cortisol. These results are in contrast to earlier studies which show that the maximal induction of a putative 19 kDa casein, α-lactalbumin and β-lactoglobulin in the tammar mammary gland is dependent upon prolactin alone. The expression of the latter three genes is not modulated by other hormones known to play a role in the in-vitro initiation of lactation in eutherians.
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ABSTRACT
The envelope protein, gp52, of the mouse mammary tumour virus (MMTV) binds to a cell-surface receptor as a first step in infection. A protein with characteristics of this receptor was measured on freshly isolated cells using, as ligand, 125I-labelled gp52 purified from C3H/HeN mice. The gp52-binding protein was found in all mouse tissues examined, but was present at highest concentrations in the mammary gland and spleen where it reached 4.2±0.3 (s.e.m.) pmol/mg protein; the dissociation constant was 30±7 pm. Binding to mammary epithelium could be displaced by either the RIII or 34I-R strains of MMTV, and binding was blocked by antibodies to gp52. Levels in the liver and adrenal glands were only 25% of those in the mammary gland, while the concentrations in the ovary and salivary gland were intermediate. Scatchard analyses of the binding data suggested that there was only a single set of high-affinity binding sites. During late pregnancy and lactation, receptor levels in mammary epithelium rose threefold, while those in the liver and salivary gland were unchanged. This induction would result in the mammary gland having 12 times the gp52-binding protein than other tissues and may result in the preferential reinfection of this tissue during lactation, with subsequent tumour formation.
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Pregnancy is characterized by increased erythropoiesis within maternal and fetal compartments. The placenta has been shown to produce factors that stimulate erythropoiesis but convincing evidence for placental production of erythropoietin (EPO) is still lacking. Prolactin-like protein E (PLP-E) was recently found to stimulate expression of the adult beta major globin gene in mouse erythroleukemia cells. Here we demonstrate that PLP-E transiently expressed in COS-7 cells stimulates proliferation and erythroid differentiation of murine and human erythroid progenitor cell lines. Electrophoretic mobility shift assays were used to show the activation of STAT5 by PLP-E in the human erythroid cell line TF1. Furthermore, we compared the effects of PLP-E on murine myeloid FDCP1 cells which do not express EPO receptors (EPORs) with effects on cells genetically engineered to express functional EPORs. We provide evidence that PLP-E-dependent proliferation and STAT5 activation is independent of the expression of the EPOR. Taken together, these data suggest that PLP-E acts on specific receptors of erythroid-committed murine and human cells by the activation of intracellular signaling pathways promoting cell growth and differentiation.
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In the common brushtail possum (Trichosurus vulpecula) lactation lasts for 200 days and consists of two distinct phases. Milk composition changes dramatically between phase 2 and 3, which correspond to early and late lactation respectively (phase 1 corresponds to pregnancy). RNA expression patterns have been established for eight major milk protein genes throughout lactation in possum mammary glands. The levels of mRNA expressed from two genes, encoding the early and late lactation proteins, were differentially regulated during lactation, with peak RNA levels occurring in phase 2 and 3 of lactation respectively. Expression of these two RNA transcripts did not overlap, and neither gene was expressed at significant levels between days 116 to 125, suggesting that the transition from phase 2 to phase 3 of lactation occurs at this time. The level of lysozyme, alpha-lactalbumin and trichosurin mRNA increased in phase 3 of lactation, whereas the levels of beta-lactoglobulin, alpha-casein and beta-casein mRNA remained constant throughout lactation. In the non-suckled gland, expression of milk protein genes was greatly reduced by day 6 of lactation. In conclusion, the early and late lactation protein genes are good markers for phase 2 and 3 of lactation, with the transition between these phases occurring around day 120 of lactation in the possum.
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ABSTRACT
During pregnancy, a placental calcium pump maintains the fetus in a hypercalcaemic state relative to the mother, a condition which has been thought to facilitate normal development of the fetal skeleton. Based on experiments performed in the sheep, parathyroid hormone-related protein (PTHrP) has been implicated as the hormone responsible for maintaining the placental calcium pump. In the present study on mice in which the PTHrP gene has been ablated by homologous recombination, we have measured both fetal and maternal circulating total and ionised calcium levels, as well as fetal total body calcium, in order to determine whether absence of PTHrP during fetal development has an effect on fetal calcium levels. Our results show that, in fetuses lacking PTHrP, circulating ionised calcium levels are significantly lower than those of heterozygote and wild-type littermates, but circulating total calcium levels show no difference. Total body calcium levels of null mutants are significantly higher than those of normal littermates.
The role of PTHrP in maintaining the integrity of the transplacental calcium pump in the rodent thus remains unclear. It may be that the lower levels of fetal blood ionised calcium in mutant animals are due to disruption of the placental pump, but, if this is the case, compensatory mechanisms have operated to allow the excessive calcium deposition seen in the skeletons of these animals. Alternatively, the increased avidity of the bones for calcium may in itself have produced a lower equilibrium level of available ionised calcium.
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Adrenomedullin (AM) is a recently identified amidated peptide produced by a variety of tissue types. We have investigated the involvement of AM and its receptor (AM-R) in developing mouse mammary glands and have examined what influence ovarian hormones have on AM and AM-R expression in this system. Tissues from ductal morphogenesis, virgin adult, pregnancy, and lactation stages were assessed for AM and AM-R by molecular, biochemical and immunohistochemical techniques. Results from these studies indicated that messenger RNA for AM and AM-R and immunoreactivity for AM were expressed in the luminal epithelium of small and large ducts and in terminal end buds. Immunoreactive AM was identified as a cytoplasm component of ductal cells, with some cells also having nuclear staining. Western blot analysis of mammary gland tissues yielded two molecular mass species (M(r) 14,000 and 18,500) of AM immunoreactivity in the mammary gland for the above developmental stages, consistent with processed intermediate and prohormone forms respectively. Ovariectomy alone or followed by hormonal treatments did not alter the expression pattern for these two proteins. By Western blot, the fully processed AM form (M(r) 6000) was identified in milk extracts from lactating glands. These data suggest a potential role for AM and its receptor in the maintenance of mammary gland homeostasis and suggests a potential role for AM in development of the newborn.
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ABSTRACT
Relaxin is a hormone associated with pregnancy that relaxes uterine smooth muscle and softens the connective tissues of the cervix and pelvis. In spite of these well-characterized tissue responses, the second messenger system linked to the relaxin receptor and the range of target tissues are only modestly understood. We found that relaxin enhanced the cyclic AMP levels in anterior pituitary cells from adult female rats. Relaxin induced a maximal 5·7±0·5-fold (mean±s.e.m.) stimulation of cyclic AMP accumulation and had an excitatory concentration for half maximal effect (EC50) of 0·4±0·1 nm, while human relaxin A and B chains had no such activity (EC50> 1 μm). Pertussis toxin amplified the efficacy of relaxin by 1·5±0·1-fold, indicating the intervention of a G coupling protein. The response to relaxin was reversible with washing, and desensitized slowly with continuous exposure to relaxin.
In an attempt to define the physiological role for relaxin at the anterior pituitary, we found that two of the major hypophysiotrophic hormones of the brain (dopamine and somatostatin) markedly inhibited the relaxin stimulation of cyclic AMP. There was also a significant correlation of the response magnitude with the gender of the donor rat. Anterior pituitary cells from adult males exhibited a mean twofold maximal stimulation after relaxin, compared with the sixfold increase measured in cells from female rats. We hypothesize a novel physiological function of relaxin, that of signalling the feminine anterior pituitary.
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We have used quantitative RT-PCR to analyse the mRNA expression profile of the major components of the IGF axis in different stages of murine mammary gland development, including late pregnancy, lactation and involution. We have shown that all the genes studied, IGF-I, IGF-II, IGF receptor (IGFR) and IGF-binding protein (IGFBP)-1 to -6, were expressed in every stage, albeit at greatly differing levels and displaying unique expression profiles between developmental stages. IGF-I was always expressed at significantly higher levels than either IGF-II or IGFR. This suggests that IGF-I may be the more important IGF during mammary morphogenesis. Overall, IGFBP-3 demonstrated the highest level of expression of any of the IGFBP genes throughout all the developmental stages studied. However, within developmental stages, by far the highest level of expression of any of the IGFBPs was that of IGFBP-5 at day 2 of involution; this was almost an order of magnitude higher than any of the other IGFBP levels recorded. This corroborated our previous findings that the levels of IGFBP-5 protein are highly elevated in the involuting mammary gland, and demonstrated that this up-regulation of IGFBP-5 operates at the level of transcriptional control or message stability. Comparison of the expression profile for these different genes would strongly suggest that they are likely to have differential functions throughout mammary gland development, and also highlights potential interactions and co-regulation between different members of this axis. In addition, our results have identified some similarities and differences in the expression of IGFBPs between the mouse mammary epithelial cell line, HC11, and the normal mammary gland which are worthy of study, most notably the differential regulation of IGFBP-2 and the site of expression of IGFBP-4 and -6. Overall, this study has demonstrated the importance and complexity of the IGF axis during mammary gland development and provides a valuable resource for future research in this area.
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The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.
