The Epithelial Glucocorticoid Receptor Is Required for the Normal Timing of Cell Proliferation during Mammary Lobuloalveolar Development but Is Dispensable for Milk Production
Tim M. Wintermantel,
Dagmar Bock,
Verena Fleig,
Erich F. Greiner and
Günther Schütz
Molecular Biology of the Cell I, German Cancer Research Center, D-69120 Heidelberg, Germany
Address all correspondence and requests for reprints to: Professor Dr. Günther Schütz, Molecular Biology of the Cell I, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: g.schuetz{at}dkfz.de.
 |
ABSTRACT
|
---|
Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and ß-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.
 |
INTRODUCTION
|
---|
GLUCOCORTICOIDS ARE INVOLVED in numerous physiological processes, among them the regulation of growth, metabolic homeostasis, and immune response. They exert their biological activity via binding to their cognate receptors, namely the glucocorticoid and mineralocorticoid receptors [glucocorticoid receptor (GR), NR3C1; and mineralocorticoid receptor (MR), NR3C2].
Glucocorticoids have long been known to act as lactogenic hormones in cell and tissue culture of mammary epithelia. Several studies have demonstrated a synergistic effect of glucocorticoids and prolactin on mammary epithelial cell differentiation and milk protein production (reviewed in Ref.1). From studies done in mammary epithelial cell culture, it has been suggested that this lactogenic activity might be due to a stimulatory effect of the GR on milk protein gene transcription. This action may involve a ligand-dependent interaction of the GR with the prolactin-activated transcription factor STAT5 (signal transducer and activator of transcription 5), leading to enhanced transcription of the ß-casein gene (2). The physiological significance of this cross-talk for mammary epithelial cell function, however, is not known.
Using gene-targeting techniques, researchers have been able to define the role of several signaling molecules in mammary gland development [reviewed by Hennighausen and Robinson (3)]. Mice lacking the GR in all tissues die at birth and, therefore, their use is limited in the investigation of mammary gland function (Refs.4 and 5 and see below). To investigate specific GR functions in mammary gland development, mice harboring a point mutation in the GR gene (GRdim) were analyzed (6). This mutation abolishes DNA-binding-dependent transcriptional regulation by GR but does not affect transcriptional regulation mediated by protein-protein interactions of GR with other transcription factors. These GRdim mice are viable and fertile (7). Mammary gland development in these mice is impaired due to a reduced ductal epithelial cell proliferation in virgin mice. Pregnancy-associated mammary gland development and lactation, however, remain normal in GRdim mice. These results suggested a new function for the GR in mammary gland development, i.e. the control of ductal epithelial cell proliferation in the virgin animal. Therefore, GR was assumed to exert its functions in lobuloalveolar differentiation and milk protein gene expression by GR-STAT5 interaction, which is not impaired in GRdim mice. Using the GRdim mouse model, however, not all functions of the GR molecule in mammary gland development can be dissected.
In a different experimental approach, embryonic mammary gland anlagen of GR-deficient mice (4) was transplanted into the cleared mammary fat pad of syngeneic hosts. Kingsley-Kallesen et al. (8) demonstrated aberrant ductal morphogenesis in transplants of GR-deficient animals, but no defect in cell proliferation, and normal lobuloalveolar development during pregnancy and lactation. Milk protein gene expression was normal in these mice, and the authors suggested that MR could compensate for GR in GR-deficient transplants. They demonstrated that, in explant cultures, the MR ligand fludrocortisone is able to stimulate ß-casein expression. Interpretation of these results may be complicated by two facts: First, although the mammary transplant method is an acknowledged experimental procedure by which to study ductal and lobuloalveolar development, it is not possible to observe a period of lactation and milk production under normal circumstances. Second, it could not be ruled out that the GR-deficient mice used as transplant donors in Ref.8 had residual GR activity, as they had been derived from a mouse line that carries a GR hypomorph allele (4, 9).
To define the biological function of the GR in the adult, intact mammary gland, we generated a mouse model with a mammary-epithelial specific ablation of the GR gene in the adult mouse using the Cre-loxP recombination system. Using a conditional allele for the GR (10) and a transgenic mouse line expressing the Cre recombinase in secretory epithelial cells of the lactating mammary gland (11), we were able to obtain mice in which the GR gene was deleted in the late stages of pregnancy, leading to loss of epithelial GR in mammary glands throughout lactation. In these mice, we analyzed lobuloalveolar morphogenesis, epithelial cell proliferation, and milk protein production.
 |
RESULTS
|
---|
The GR Gene Is Deleted in Alveolar Epithelial Cells of GRloxP/loxP; WAPiCre Mice, Leading to a Loss of GR Protein from the Mammary Epithelium during Late Pregnancy
We have shown previously that the WAPiCre is active in pregnant mammary glands, leading to complete deletion of loxP-flanked genes by lactation d 3 (L3) (11). To analyze the kinetics of the inactivation of the GR gene, we analyzed DNA from mammary gland tissue of GRloxP/loxP; WAPiCre mice and assayed recombination of the GR gene by Southern blot analysis (Fig. 1
). We found elevated levels of mammary-specific recombination from d 14.5 of pregnancy (P14.5). Recombination levels reached 60% at L0.5 and remained at 65% during lactation. In line with our previous observations using WAPiCre mice (11), this suggests almost complete recombination of the GR allele in the epithelial compartment, which accounts for about 70% of total cells in the lactating mouse mammary gland. The faint bands seen in the virgin glands correspond to non-tissue-specific background recombination in the WAPiCre mice that we reported earlier (11).

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 1. Southern-Blot Analysis of Recombination in Mammary Gland DNA
DNA was isolated from mammary glands of GRloxP/loxP; WAPiCre mice in several stages of mammary gland development (as indicated above): virgin, P14.5, P16.5, P18.5, L0.5, L10. DNA was cleaved with SacI and hybridized with a GR-specific probe to detect floxed (7 kb) and deleted (4.8 kb) alleles. Mammary gland DNA from a control GRloxP/loxP mouse in the absence of the transgene (lane labeled c) shows only floxed band. Recombination percentages are given as average of two lanes.
|
|
Next, we investigated whether the ablation of the GR gene leads to a loss of GR protein at the onset of lactation. Figure 2
, A and B, shows that GR is present in ductal epithelial cells of virgin mammary glands of all genotypes, consistent with the absence of WAPiCre activity at this stage of mammary gland development. At P14.5, GR can be detected in the developing alveolar lobes of control mice (Fig. 2C
), but is lacking the majority of epithelial cells in mutant animals (Fig. 2D
). These results suggest that the loss of GR takes place after the initiation of lobuloalveolar development. At the onset of lactation, GR is present in all epithelial cell nuclei of control mammary glands (Fig. 2E
), but absent from the vast majority of epithelial cells in the mutant glands (Fig. 2F
). We could not detect any GR-positive epithelial cell nucleus in mutant glands at L2 and later (Fig. 2
, panel H vs. panel G). This suggests that GR inactivation is complete in the epithelial cells of the lactating mammary glands of GRloxP/loxP; WAPiCre mice. In contrast, stromal cells retain GR protein during all stages of mammary gland development in mutant animals (see, for example, Fig. 2H
).

View larger version (111K):
[in this window]
[in a new window]
|
Fig. 2. GR Protein Is Lost in Mammary Glands of GRloxP/loxP; WAPiCre Mice
Immunohistochemical detection of GR (brown 3,3'-diaminobenzidine staining) in mammary glands of virgin (A and B), P14.5 (C and D), L0.5 (E and F) and L3 (G and H), control (A, C, E, and G) and GRloxP/loxP; WAPiCre (B, D, F, and H) mice. Note that mammary epithelium is GR positive in virgin mutant mice, partly GR negative at P14.5 and completely devoid of GR at L3. Scale bar, 250 µm.
|
|
Loss of GR Does Not Critically Impair Mammary Gland Function during Lactation
Female mice lacking GR in the secretory epithelium are able to nurse their pups. However, in litters from GRloxP/loxP; WAPiCre-mice, pup weights during the first 10 d of life are consistently smaller by about 20% than in control mice, independent of the genotype distribution in the litters (Fig. 3
). Next, we analyzed the histology of the GR mutant mammary glands. In GRloxP/loxP; WAPiCre-mice, secretory alveoli are formed and contain epithelial cells just as in control mice; however, less space is occupied by epithelial structures, and more space is occupied by fat compared with controls (Fig. 4
, AH). This difference is pronounced at the time of pup delivery (Fig. 4
, panel B vs. panel A), but persists throughout lactation (d 10: see Fig. 4F
and 4H
vs. 4E and 4G).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3. Pup Weight Is Reduced in Litters from GRloxP/loxP; WAPiCre Mice Compared with Pups from Control Mice
In litters that were adjusted to a litter size of eight pups/mother, pup weights were determined at L2, L5, and L10. At all time points, average pup weight in litters from control mice (open bars) was higher than in litters from GRloxP/loxP; WAPiCre mice (hatched bars), the latter value being about 80% of the former value at all time points. Plotted is the average weight ± SD. P < 0.05 for all time points according to Students t test.
|
|

View larger version (108K):
[in this window]
[in a new window]
|
Fig. 4. Alveolar Lobes Are Present, but Do Not Completely Penetrate the Fat Pad in GRloxP/loxP; WAPiCre Mammary Glands
Hematoxylin-eosin staining of control (A, C, and E) and GRloxP/loxP; WAPiCre (B, D, and F) mice during lactation: L0.5 (A and B), L3 (C and D), L10, low magnification (E and F). Whole-mount pictures of mammary glands from control (G) and GRloxP/loxP; WAPiCre (H) mice at L10 show dense alveolar structures in control mice and less dense structures in mammary gland-specific GR mutants. Scale bar, 250 µm.
|
|
Alveolar Development Is Retarded in GR Mutant Mammary Glands
To define the first time point when GR deficiency in the epithelium leads to a visible alteration in mammary gland structure, we analyzed mammary glands at P14.5, P16.5, and P18.5. Comparison between mutant and control mice revealed no alteration in mammary gland structure at P14.5 (Fig. 5
, A and B). At P16.5, however, there is a clear difference between GR-deficient (Fig. 5D
) and control (Fig. 5C
) glands in that lobuloalveolar development in GR-deficient glands has not proceeded as far as in the control glands. Analysis of mammary glands from P18.5 (Fig. 5
, E and F) and L0.5 (see Fig. 4
, A and B) shows that lobuloalveolar development is not completely blocked, but is significantly retarded in GR mutant mammary glands, enabling some alveolar structures to be observed.

View larger version (177K):
[in this window]
[in a new window]
|
Fig. 5. Alveolar Development Is Drastically Retarded in GRloxP/loxP; WAPiCre Mammary Glands during Late Pregnancy
Hematoxylin-eosin staining of control (A, C, and E) and GRloxP/loxP; WAPiCre (B, D, and F) mice during several stages of late lobuloalveolar development: P14.5 (A and B), P16.5 (C and D), and P18.5 (E and F). Scale bar, 250 µm.
|
|
Analysis of Proliferation and Apoptosis in GR Mutant Mammary Glands Reveals Altered Kinetics of Epithelial Cell Proliferation in GRloxP/loxP; WAPiCre-Mice
We next tested whether the retarded lobuloalveolar development was due to impaired proliferation or increased apoptosis. Proliferating cells were detected by bromodeoxyuridine (BrdU) labeling in both mutant and control mammary glands (Fig. 6
). At P14.5, however, less proliferating cells are seen in GR-deficient glands than in control glands (Fig. 6
, panels B and D vs. panels A and C). At this stage, we determined the mitotic labeling index (BrdU-positive cell nuclei/ total epithelial nuclei) in wild-type glands to be 8.4%, whereas in the mutants, it was 5.2%, indicating that epithelial cell proliferation is affected by GR loss.

View larger version (164K):
[in this window]
[in a new window]
|
Fig. 6. Reduced Epithelial Proliferation at P14.5, but Increased Proliferation after Parturition in GRloxP/loxP; WAPiCre Mammary Glands
Immunohistochemical detection of BrdU in proliferating cells (brown 3,3'-diaminobenzidine staining) in mammary glands of P14.5 (AD) and L0.5 (E and F) control (A, C, and E) and GRloxP/loxP; WAPiCre (B, D, and F) mice. Scale bar, 250 µm.
|
|
When examining proliferation of epithelial cells in the lactating glands, we noticed a dramatic increase in cell proliferation in GR mutant mammary glands at L0.5 (Fig. 6F
, labeling index 33% vs. 6E, labeling index 2.1%). This proliferative burst seems to compensate, in the initial stages of lactation, for the poor alveolar structures in GR mutant glands before parturition. We did not observe, however, increased apoptosis in mutant glands at any time point during pregnancy. Apoptotic cells can be observed only at the time when the epithelial expansion into the fat pad ceases, i.e. at L0.5 in control animals and at L2 in mutant glands (data not shown).
Milk Protein Expression Is Normal in GR Mutant Mammary Glands
To investigate epithelial cell differentiation and function in mammary glands devoid of GR, expression of the two milk protein genes, ß-casein and whey acidic protein (WAP), were analyzed in mutant mammary glands. Northern blot analysis shows that mRNAs for both milk proteins are expressed in GR mutant mammary glands at levels comparable to controls (Fig. 7
). We conclude that GR is not essential for the expression of milk protein genes in the lactating mammary epithelium in vivo. An estimation of milk fat percentage, by creamatocrit measurement, and a fatty acid profile did not give any evidence that fat mobilization into milk was affected by the GR loss (data not shown). After weaning, involution proceeds normally, and GRloxP/loxP; WAPiCre-mice were able to go through successive rounds of pregnancy and lactation, albeit with the same alveolar phenotype in later lactational periods as in the first one (data not shown). These results indicate that epithelial GR is not essential for mammary gland function once secretory alveoli have formed.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 7. Milk Protein Expression in GRloxP/loxP; WAPiCre Mammary Glands
Northern blot analysis reveals that ß-casein and WAP mRNA are expressed almost at wild-type levels in GR-deficient mammary glands. Total RNA was prepared, separated on a denaturing formamide/agarose gel, and blotted to a nylon membrane. Blots were hybridized either with a ß-casein or WAP-specific probe.
|
|
 |
DISCUSSION
|
---|
We have shown previously that the WAPiCre transgene catalyzes efficient cell type-specific recombination of loxP-flanked target genes, leading to complete deletion of such a target gene in mammary epithelium at L3 (11). Here, we report that, in mammary glands from GRloxP/loxP; WAPiCre mice, the floxed GR allele is deleted from alveolar epithelial cells already in late pregnancy. We observe a distinct mammary phenotype just before and after parturition although we could detect some GR-positive epithelial cells before L3 (Fig. 2
). Most of the alveolar cells lack GR protein during the whole period of observation (i.e. from P14.5 onward); therefore, the GRloxP/loxP; WAPiCre mice can serve as a tool with which to identify GR-dependent functions in the mammary gland after P14.5.
Because GRloxP/loxP; WAPiCre mice retain GR in the mammary ductal epithelium of the nonpregnant, nonlactating mouse, possible roles of GR in early mammary (ductal) development cannot be identified with this mouse model. Yet such a role can be inferred from previous work on GRdim mice that are deficient in GR binding to DNA and display altered ductal development (7).
We have shown that, in the absence of GR, mammary alveolar development is retarded. Between P14.5 and P18.5, alveoli fail to develop fully and penetrate the fat pad in GRloxP/loxP; WAPiCre. This phenotype results from impaired cell proliferation but not from increased apoptosis, as no apoptotic epithelial cells can be detected in this phase of mammary development (data not shown). Although these observations concentrate on a different time window in mammary gland development, they are similar to the findings in GRdim mice by Reichardt et al. (6). In this report, a defect in ductal morphogenesis in GRdim mice was observed that was also due to reduced epithelial cell proliferation. However, GRdim can fully support alveolar proliferation, and the structure of the lactating gland is normal in GRdim mice. Therefore, the GR-signaling pathways involved in the control of cell proliferation in the developing ducts seem to be different than those that influence cell proliferation in the alveoli. From the data on GRdim mice and our observations, we conclude that alveolar proliferation in late pregnancy is dependent on GR via a pathway that is independent from dimeric GR-glucocorticoid response element interaction. The molecular mechanism for GR influence on alveolar cell proliferation between P14.5 and P18.5 remains to be elucidated. The serum- and glucocorticoid-induced kinase has been described as a GR target that mediates proliferative and antiapoptotic effects of corticosteroids on cultured mammary epithelial cells (12). Serum- and glucocorticoid-induced kinase was detected at equal levels in mutant and control glands at P14.5 (data not shown).
After parturition, however, a dramatic increase in epithelial cell proliferation takes place in mammary glands from GRloxP/loxP; WAPiCre mice (Fig. 6
, panel E vs. panel F). This proliferation is compensating for the poor alveolar structures in mutant glands. It should be noted that this proliferative boost cannot be derived from the residual GR-positive epithelial cells, as the number of BrdU-positive epithelial cells (i.e. cells that have undergone S phase within 2 h before death) at L0.5 vastly exceeds the number of residual GR-positive epithelial cells at L0.5 or P18,5. (compare Fig. 6F
with Fig. 2F
). Therefore, compensatory mechanisms seem to take place in the absence of GR that restore proliferative capacities upon GR-negative epithelial cells. They do not suffice, however, to restore the penetration of the mammary fat pad by alveolar structures to wild-type levels. Mammary glands from GRloxP/loxP; WAPiCre mice at L10 still can easily be distinguished from wild-type glands by a decrease in alveolar space relative to fat (Fig. 4
, panels E and G vs. panels F and H). The dramatic boost of cell proliferation at L0.5 could be linked to the transition of the mammary epithelium from stage I to stage II lactogenesis, triggered by the decrease in circulating progesterone levels after parturition (13). This process has been demonstrated to rely on basal levels of glucocorticoids. As the morphological changes from pregnancy to lactation proceed normally in the absence of epithelial GR, targets for glucocorticoid actions in this process are the mammary stromal cells or endocrine cells outside of the mammary gland.
After parturition, alveolar morphology and milk production are normal in GRloxP/loxP; WAPiCre mice, as judged from histology, from monitoring milk protein gene expression and from the ability of GRloxP/loxP; WAPiCre mothers to nurse their pups. Furthermore, there was no evidence of impaired mobilization of fat into milk in GRloxP/loxP; WAPiCre mice. GR deletion does not impinge on the expression of the milk proteins, ß-casein and WAP, thus indicating that GR is not essential for ß-casein and WAP expression in vivo. The costimulatory action of liganded GR on the prolactin-activated transcription factor STAT5 in the activation of the ß-casein promotor that has been described in several cell culture model systems (2, 14) seems to be dispensable for milk protein synthesis in vivo.
Kingsley-Kallesen et al. (8) have studied epithelial transplants from mice carrying a hypomorphic allele of the GR (4, 5). They observed altered ductal development, but normal lobuloalveolar development and lactation. They suggested that the MR might compensate for the GR in milk protein synthesis. Because we cannot detect MR in mammary glands of GRloxP/loxP; WAPiCre or control mice by immunohistochemistry (data not shown), we have no data supporting a compensation of GR loss by MR in our experimental system. The MR expression observed in transplanted mammary epithelial cells by Kingsley-Kallesen et al. (8) could be an experimental feature (mouse strain, transplantation).
Kingsley-Kallesen et al. (8) showed unchanged lobuloalveolar development in the absence of GR at P14.5 and at L1. Clearly, the conditional mutagenesis model described here is different from transplantation experiments, i.e. in the latter, epithelial cells and their progenitors have been devoid of GR since zygote formation of the donor mice, and compensatory mechanisms with regard to cell proliferation could be established in these cells early on. In the conditional mutagenesis model described here, GR is deleted in mammary epithelial cells only during lobuloalveolar development, and a phenotype is seen in these cells soon after.
In addition, both studies differ in the GR allele used. Kingsley-Kallesen et al. (8) used epithelia from mice carrying a hypomorphic GR allele (4) that retain some activity (9). The conditional GR allele in our study involves a different targeting strategy, which leads to a nonfunctional GR allele after deletion (5, 10).
Apart from these seeming discrepancies, it should be noted that the observations reported here, by and large, corroborate those of Kingsley-Kallesen et al. (8) with respect to lactation, milk protein expression, and involution proceeding normally in the absence of GR. Corticosteroids have been shown to regulate the postlactational involution of the mammary gland by inhibiting the synthesis of matrix metalloproteinases (15). This process probably occurs in cells of the mammary stroma, which remain GR positive in both the cell type-specific deletion model presented here and in the epithelial transplantation model presented by Kingsley-Kallesen et al. (8).
In summary, our observations show that GR regulates mammary epithelial cell proliferation during late lobuloalveolar development, but is dispensable for the differentiation and function of secretory alveolar cells of the lactating mammary gland. As the initiation of lobuloalveolar development takes place in the presence of GR in the model presented here, it may be rewarding, in further studies toward glucocorticoid signaling in the mammary gland, to target GR in ductal epithelial cells or alveolar stem cells. As demonstrated here, loss-of-function experiments can be performed in a highly specific manner in vivo using the Cre-loxP system, once appropriate molecular markers for the respective cell type are identified.
 |
MATERIALS AND METHODS
|
---|
Animals
Mice with a conditional allele of the GR have been described [GRloxP mice (10)]. In these mice, exon 3 of the GR is flanked by loxP sequences. Deletion of this exon, which codes for the first zinc finger of the GR DNA binding domain, leads to a nonfunctional GRnull allele. A transgenic mouse line expressing the Cre recombinase in secretory epithelial cells of the lactating mammary gland has also been described [WAPiCre mice (11)]. These two mouse lines have been bred on a FVB/N background, and crossed to produce mice homozygous for the GRloxP allele and transgenic for the WAPiCre, with both heterozygous GRloxP/+;WAPiCre mice and homozygous GRloxP/loxP nontransgenic mice serving as controls. All animal experiments were performed at the German Cancer Research Center according to institutional and international standards.
Analysis of Recombination
DNA was prepared from mouse mammary glands by overnight lysis in tail buffer (50 mM Tris HCl, pH 8.0; 100 mM EDTA; 100 mM NaCl; 1% sodium dodecyl sulfate; 0.5 mg/ml proteinase K) and subsequent phenol-chloroform extraction; 1030 µg of organ DNA were digested with SacI, blotted, and hybridized with a probe to detect the floxed (7 kb) and recombined (4.8 kb) alleles. Signals were quantified using a Phospho Imager (Fuji, Tokyo, Japan). Recombination percentage is calculated as signal intensity of the band representing the recombined allele divided by the sum of the signal intensities of the bands representing recombined and floxed alleles.
Immunohistochemistry and BrdU Labeling
Mice were mated at 8 wk of age and killed on the respective stages of mammary gland development by cervical dislocation. Two hours before death, a solution of BrdU in PBS was injected at 50 mg BrdU/kg body weight. Inguinal mammary glands were removed, fixed overnight in 4% PBS-buffered paraformaldehyde, dehydrated, and embedded in paraffin, and 6-µm sections were prepared. Antigens were detected using specific antibodies diluted in PBS [GR: MC-20, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); MR: rabbit polyclonal antibody (16); BrdU: DAKO M0744 (DAKO Corp., Carpinteria, CA] and visualized by ABC/peroxidase staining (Vectastain; Vector Laboratories, Inc., Burlingame, CA). To visualize nuclei, sections were stained with hematoxylin after immunodetection. Mitotic labeling index has been determined by counting at least 500 epithelial cell nuclei per animal and determining the number of BrdU-positive nuclei.
RNA Isolation and Analysis
RNA from snap-frozen tissue was isolated using the RNeasy mini kit (QIAGEN, Chatsworth, CA) according to manufacturers instructions. Total RNA (15 µg) was run on a denaturing formamide-3[N-morpholino]propanesulfonic acid-1% agarose gel, transferred to a nylon membrane, and hybridized to a probe labeled with
32P-dCTP by random priming. Probe templates were generated by restriction digestion from cDNA fragments subcloned into plasmids, namely pFLAG-ß-casein (mouse), kindly provided by Dr. N. Hynes, Friedrich-Miescher-Institute, Basel (CH), pEndoB (mouse keratin 18), kindly provided by Dr. W. Franke, German Cancer Research Center, Heidelberg (D), and an expressed sequence tag-cDNA probe for WAP (IMAGE clone 1511486). Signals were detected and quantified using a PhosphoImager.
Milk Fat Analysis
Milk was isolated from mice at L5. Six hours after pup removal, 25 IU oxytocin in PBS was injected ip, and 15 min later, milk was collected by aspiration in a Pasteur pipette. Fat percentage was estimated using the creamatocrit method (17). A fatty acid profile from milk was determined by gas chromatography/mass spectometry (18, 19).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Stefan Berger for helpful advice on MR immunohistochemistry; Drs. Nancy Hynes, Jeffrey M. Rosen, and Lothar Hennighausen for helpful discussions; and Dr. Jürgen Okun and Patrik Feyh, Heidelberg University Center for Metabolic Disease/Metabolic Laboratory, for the analysis of fatty acids from mouse milk.
 |
FOOTNOTES
|
---|
Present address for E.G.: Evotec OAI AG, Schnackenburgallee 114, D-22525 Hamburg, Germany.
First Published Online October 7, 2004
Abbreviations: BrdU, Bromodeoxyuridine; GR, glucocorticoid receptor; L3, lactation d 3; MR, mineralocorticoid receptor; P14.5, pregnant d 14.5; STAT5, signal transducer and activator of transcription 5; WAP, whey acidic protein.
Received for publication February 17, 2004.
Accepted for publication September 30, 2004.
 |
REFERENCES
|
---|
- Rosen JM, Zahnow C, Kazansky A, Raught B 1998 Composite response elements mediate hormonal and developmental regulation of milk protein gene expression. Biochem Soc Symp 63:101113[Medline]
- Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional interactions between Stat5 and the glucocorticoid receptor. Nature 383:726728[CrossRef][Medline]
- Hennighausen L, Robinson GW 2001 Signaling pathways in mammary gland development. Dev Cell 1:467475[Medline]
- Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G 1995 Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 9:16081621[Abstract]
- Tronche F, Kellendonk C, Reichardt HM, Schütz G 1998 Genetic dissection of glucocorticoid receptor function in mice. Curr Opin Genet Dev 8:532538[CrossRef][Medline]
- Reichardt HM, Horsch K, Grone HJ, Kolbus A, Berg H, Hynes N, Schutz G 2001 Mammary gland development and lactation are controlled by different glucocorticoid receptor activities. Eur J Endocrinol 145:519527[Medline]
- Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R, Gass P, Schmid W, Herrlich P, Angel P, Schutz G 1998 DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93:531541[Medline]
- Kingsley-Kallesen M, Mukhopadhyay SS, Wyszomierski SL, Schanler S, Schutz G, Rosen JM 2002 The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development. Mol Endocrinol 16:20082018[Abstract/Free Full Text]
- Mittelstadt PR, Ashwell JD 2003 Disruption of glucocorticoid receptor exon 2 yields a ligand-responsive C-terminal fragment that regulates gene expression. Mol Endocrinol 17:15341542[Abstract/Free Full Text]
- Tronche F, Kellendonk C, Kretz O, Gass P, Anlag K, Orban PC, Bock R, Klein R, Schutz G 1999 Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet 23:99103[CrossRef][Medline]
- Wintermantel TM, Mayer AK, Schutz G, Greiner EF 2002 Targeting mammary epithelial cells using a bacterial artificial chromosome. Genesis 33:125130[CrossRef][Medline]
- Mikosz CA, Brickley DR, Sharkey MS, Moran TW, Conzen SD 2001 Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1. J Biol Chem 276:1664916654[Abstract/Free Full Text]
- Neville MC, Morton J 2001 Physiology and endocrine changes underlying human lactogenesis II. J Nutr 131:3005S3008S
- Doppler W, Windegger M, Soratroi C, Tomasi J, Lechner J, Rusconi S, Cato AC, Almlof T, Liden J, Okret S, Gustafsson JA, Richard-Foy H, Starr DB, Klocker H, Edwards D, Geymayer S 2001 Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5. Mol Cell Biol 21:32663279[Abstract/Free Full Text]
- Lund LR, Romer J, Thomasset N, Solberg H, Pyke C, Bissell MJ, Dano K, Werb Z 1996 Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development 122:181193[Abstract/Free Full Text]
- Kretz O, Schmid W, Berger S, Gass P 2001 The mineralocorticoid receptor expression in the mouse CNS is conserved during development. Neuroreport 12:11331137[CrossRef][Medline]
- Lucas A, Gibbs JA, Lyster RL, Baum JD 1978 Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk. Br Med J 1:10181020[Medline]
- Loor JJ, Lin X, Herbein JH 2003 Effects of dietary cis 9, trans 1118:2, trans 10, cis 1218:2, or vaccenic acid (trans 1118:1) during lactation on body composition, tissue fatty acid profiles, and litter growth in mice. Br J Nutr 90:10391048[CrossRef][Medline]
- Moser HW, Bergin A, Cornblath D 1991 Peroxisomal disorders. Biochem Cell Biol 69:463474[Medline]