Pattern of expression of the KGF receptor and its ligands KGF and FGF-10 during postnatal mouse mammary gland development

The expression of the KGF receptor (KGFR) and its stromal ligands, KGF and FGF-10, was compared during mouse mammary gland development. KGFR expression in mammary parenchyma is maximal in mature virgin mice, declines during pregnancy and lactation, but rises after weaning. The rise in KGFR mRNA in the virgin animal corresponds to parenchymal growth. The fall in KGFR expression in pregnancy is driven by hormone-induced alveolar differentiation since the level of KGFR mRNA is 5-fold higher in isolated ductal cells compared to alveolar cells. KGF and FGF-10 expression patterns differ during ductal development. FGF-10 is also expressed at about a 15-fold higher molar level than KGF. During pregnancy and lactation, expression of KGF and FGF-10 decreases in intact fat pads but is unchanged in parenchyma-free fat pads. Thus, the decrease in KGF and FGF-10 expression observed in intact glands during pregnancy and lactation is not a direct consequence of the changing hormonal milieu but more likely reflects an increase in the ratio of epithelium to stroma. Differences in the level and pattern of expression of mRNA for KGF, FGF-10, and the KGFR during postnatal development of the mouse mammary gland are a result of morphological development, changes in the ratio of stroma to epithelium, and hormonal regulation of cell differentiation. These changes suggest that the biological roles that these growth factors play are regulated by fluctuations in both growth factor and growth factor receptor expression and that KGF and FGF-10 may have different regulatory functions.     

Variation in the lack of polyadenylation of the rat milk protein mRNAs during the lactation cycle

Translationally active milk protein mRNAs were found as nonpolyadenylated mRNAs in the rat mammary gland during pregnancy, lactation and involution. Analyses of whey protein mRNA and casein mRNA with the corresponding cDNAs showed that the lack of polyadenylation of these mRNAs at different time points of the lactation cycle is not consistent with the hypothesis that polyadenylation may be incomplete in the mammary gland when large amounts of mRNA are synthesized. The fraction of whey protein mRNA and casein mRNA that lacked polyadenylation was inversely proportional to the concentration of each sequence in the tissue during pregnancy, lactation and involution. A model is proposed to explain the finding that in each animal the ratio of casein mRNA to whey protein mRNA was similar in polyadenylated RNA and in nonpolyadenylated RNA at all stages of the lactation cycle.     

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.     

Milk-protein mRNAs and poly(A) in the involuting rat mammary gland return to the levels found during late pregnancy

Analyses of the rat mammary gland show that the increase in the milk-protein mRNAs during the development of lactation and the rapid disappearance of these sequences during involution are not accompanied by similar changes in the poly(A) content. During the development of lactation the casein mRNA is initially in great excess to the whey-protein mRNA and this differential expression of the genes for the two types of milk proteins is again observed during early involution. Since the amounts of poly(A) and of both milk-protein mRNAs are also similar to the amounts found in the gland during late pregnancy, these results indicate that during early involution the mammary gland has reverted to the pattern of mRNA metabolism that occurs during late pregnancy.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.     

Plasminogen activators in the mouse mammary gland. Decreased expression during lactation

The enzyme content and mRNA level for both urokinase-type and tissue-type plasminogen activators have been explored during the life cycle of the adult mouse mammary gland. Both enzymes were detected, and urokinase-type plasminogen activator was the predominant form. A marked decrease in enzyme content occurred in late gestation and was maintained throughout lactation; upon weaning, the enzyme content returned to the levels found in virgin mice. These effects were entirely accounted for by changes in the respective mRNA concentrations, which were determined with respect to both total tissue RNA and poly(A+) mRNA. Thus, plasminogen activator-catalyzed proteolysis may occur at high levels throughout the life cycle of the mouse mammary gland, except during lactation.     

Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation

Expression of the mouse whey acidic protein (WAP) gene is specific to the mammary gland, is induced several thousand-fold during pregnancy, and is under the control of steroid and peptide hormones. To study developmental regulation of the mouse WAP gene, a 7.2-kilobase (kb) WAP transgene, including 2.6 kb of 5'- and 1.6 kb of 3'-flanking sequences, was introduced into mice. Of the 13 lines of mice examined, 6 expressed the transgenes during lactation at levels between 3 and 54% of the endogenous gene. Although expression was dependent on the site of integration, the transgenes within a given locus were expressed in a copy number-dependent manner and were coordinately regulated. The WAP transgenes were expressed specifically in the mammary gland, but showed a deregulated pattern of expression during mammary development. In all six lines of mice, induction of the WAP transgenes during pregnancy preceded that of the endogenous gene. During lactation, expression in two lines increased coordinately with the endogenous gene, and in three other lines of mice, transgene expression decreased to a basal level. These data indicate that the 7.2-kb gene contains some but not all of the elements necessary for correct developmental regulation. At a functional level it appears as if a repressor element, which inactivates the endogenous gene until late pregnancy, and an element necessary for induction during lactation are absent from the transgene. Complementary results from developmental and hormone induction studies suggest that WAP gene expression during pregnancy and lactation is mediated by different mechanisms.     

Non-coordinate expression of closely linked mouse casein genes

Expression levels of five mouse casein genes were analysed in the mammary gland of virgin, pregnant and lactating mice. We have already shown that the five murine casein genes are arranged in the order, alpha-beta-gamma-epsilon-kappa in a tandem array, very close to each other in a 250 kb DNA fragment of mouse genome. Northern blot analysis showed that, of the calcium-sensitive casein genes, the epsilon casein gene is expressed only during lactation unlike the alpha, beta and gamma casein genes which are expressed during pregnancy and lactation. Even though the alpha, beta and gamma genes exhibited a co-ordinated expression pattern from mid to the later stages of pregnancy, the mRNA levels varied considerably (60, 90 and 100% respectively) by the onset of lactation. The mRNA level of the calcium-insensitive kappa casein gene increased from mid-pregnancy but at a lower rate and reached approximately 60% by the first day of lactation. Considering the locations and closeness of the casein genes, a non-coordinate expression profile is exhibited by the mouse casein genes, particularly the epsilon casein gene.     

Differential expression of TGF-β isoforms during postlactational mammary gland involution

After cessation of lactation, the mammary gland undergoes involution, which is characterized by a massive epithelial cell death and proteolytic degradation of the extracellular matrix. Whereas the expression patterns and also the function of TGF-beta isoforms during mammary gland branching morphogenesis and lactation are well understood, their expression during postlactational involution and therefore a possible role in this process is poorly known. In this study we show that TGF-beta3 expression is dramatically induced (>fivefold) during mouse mammary gland involution when compared to that of virgin mouse, reaching a maximal expression level at day 4 after weaning. In contrast, other TGF-beta isoforms do not display significant increase in expression during involution (TGF-beta1, 1.3-fold and TGF-beta2, <1.5-fold) when compared to that of virgin or lactating mice. During mammary gland involution, TGF-beta3 is expressed in the epithelial layer and particularly in myoepithelial cells. A comparison of the kinetics of TGF-beta3 expression to that of programmed cell death and degradation of the basement membrane suggests that TGF-beta3 functions in the remodeling events of the extracellular matrix during the second stage of involution.     

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands.

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.     

Cold-inducible RNA binding protein in mouse mammary gland development

RNA binding proteins (RBPs) regulate gene expression by controlling mRNA export, translation, and stability. When altered, some RBPs allow cancer cells to grow, survive, and metastasize. Cold-inducible RNA binding protein (CIRP) is overexpressed in a subset of breast cancers, induces proliferation in breast cancer cell lines, and inhibits apoptosis. Although studies have begun to examine the role of CIRP in breast and other cancers, its role in normal breast development has not been assessed. We generated a transgenic mouse model overexpressing human CIRP in the mammary epithelium to ask if it plays a role in mammary gland development. Effects of CIRP overexpression on mammary gland morphology, cell proliferation, and apoptosis were studied from puberty through pregnancy, lactation and weaning. There were no gross effects on mammary gland morphology as shown by whole mounts. Immunohistochemistry for the proliferation marker Ki67 showed decreased proliferation during the lactational switch (the transition from pregnancy to lactation) in mammary glands from CIRP transgenic mice. Two markers of apoptosis showed that the transgene did not affect apoptosis during mammary gland involution. These results suggest a potential in vivo function in suppressing proliferation during a specific developmental transition.     

Leptin mRNA expression in the rat mammary gland at different activation stages

Leptin is expressed in various tissues, suggesting that this protein is effective not only at the central nervous system level, but also peripherically. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues, but there is no information available concerning expression levels of leptin in the rat mammary gland at different activation stages. We used semi-quantitative RT-PCR to investigate leptin mRNA expression levels in the rat mammary gland at different activity stages. Rat mammary gland samples were collected from virgin females and on days 6, 12, 18 of pregnancy and of lactation (six rats per group). The expression levels of leptin mRNA were measured by semi-quantitative RT-PCR, with β-actin as an internal control. Leptin mRNA was highly expressed in virgin rat mammary glands (leptin(IOD)/β-actin(IOD) = 1.60). It decreased gradually during pregnancy, being lowest at 18 days of pregnancy, when the levels were significantly lower than in virgin mammary tissue. Leptin mRNA increased slightly during lactation, but the difference was not significant. By day 18 of lactation, expression levels of leptin mRNA reached the same values as in virgin mammary tissue (leptin(IOD)/β-actin(IOD) = 1.65). Based on these results, we suggest that leptin has an important regulation role in rat mammary gland activation.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide

RNAase H, which catalyzes the hydrolysis of the RNA moiety of an RNA-DNA hybrid, was measured in the mammary gland of virgin, pregnant, lactating, and weaning Fischer rats and in the R3230AC mammary tumor grown in the same animals. In the normal mammary gland when DNA levels were low, as in the virgin state or during involution, RNAase H activity was also low. During pregnancy and lactogenesis when DNA levels increased, RNAase H activity, either on the basis of mammary gland weight or DNA content, also increased. During lactation when cellular proliferation ceases but rates of RNA and protein synthesis continue to reach peak values, RNAase H activity decreased. Compared to the corresponding enzyme from host glands, RNAase H from the R3230AC mammary tumor grown in pregnant and lactating hosts changes similarly, but to a lesser extent. The RNAase H activity which, ona tissue weight basis, was higher than in normal tissue also increased during pregnancy and directly after parturition, but decreased during lactation. During pregnancy these changes were accompanied by an increase in tumor DNA values. During lactation the tumor DNA values returned to the level seen in virgin hosts. These results are consistent with a role for RNAase H in DNA replication in rat mammary gland and in R3230Ac mammary tumor.     

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate.

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.     

Studies of mitochondrial development prior to lactogenesis in the mouse mammary gland

Studies were performed to define mitochondrial development in relation to epithelial cell proliferation and differentiation prior to lactogenesis in the mammary gland of the mouse. Gland weight, total DNA, and total protein, selected as criteria of gland growth and proliferation, and various parameters of mitochondrial development were followed throughout the period. Gland weight and total DNA started increasing about Day 5 of pregnancy and reached maximal values by parturition. Total gland protein began to increase at the same time but did not reach maximal values until Day 4 of lactation. Total mitochondrial protein and the mitochondrial marker enzyme activities, succinate oxidase, succinate-linked ATP formation, and cytochrome oxidase increased gradually during pregnancy with rapid 2- to 3-fold increases occurring during the early days of lactation. Similarly, succinate oxidase activity per unit DNA of isolated mammary parenchymal cells increased gradually from mid-pregnancy to parturition with a precipitous, 2-fold increase occurring during early lactation.