A Short Form of the Prolactin (PRL) Receptor Is Able to Rescue Mammopoiesis in Heterozygous PRL Receptor Mice
Nadine Binart,
Prune Imbert-Bolloré,
Nathalie Baran,
Céline Viglietta and
Paul A. Kelly
Hormone Targets (N.B., P.I.-B., N.B., P.A.K.), Institut National de la Santé et de la Recherche Médicale Unit 584, Faculté de Médecine Necker-Enfants Malades, 75730 Paris, France; and Unité de Différenciation Cellulaire (C.V.), Institut National de la Recherche Agronomique, 78352 Jouy en Josas, France
Address all correspondence and requests for reprints to: Nadine Binart, Hormone Targets, Institut National de la Santé et de la Recherche Médicale, Unité 584, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris, France. E-mail: binart{at}necker.fr.
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ABSTRACT
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The heterozygous prolactin (PRL) receptor (PRLR +/-) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. The PRLR is expressed ubiquitously, with various proportions of long and short isoforms in different tissues. Conflicting data have appeared on the putative role of the receptor short forms, with both agonist and antagonistic actions proposed. To assess whether the mouse PR-1 short isoform of the PRLR is potentially able to transduce a signal, we overexpressed it in heterozygous mice and investigated its effect on the rescue of mammary development. PRLR+/- mice were not able to develop a functional mammary gland, but restoration of mammary alveolar development and an increase in the expressions of casein and whey acidic protein genes were observed in transgenic PRLR+/- mice expressing the short form of the PRLR, leading to a complete rescue of mammary gland development and function in young females. These results demonstrate that PR-1, the short form of the PRLR, can improve mammary development in PRLR+/- mice, which compensates for the haploinsufficiency of the receptor long form; this effect is probably caused by accelerated proliferation and an activation of the PRLR signaling cascade, resulting in activation of target genes involved in mammary development and milk synthesis.
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INTRODUCTION
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THE EXISTENCE OF multiple forms of the prolactin (PRL) receptor (PRLR) could explain the pleiotropic actions of PRL in a wide variety of cells. PRLRs are present in a large number of tissues, and the pattern of distribution of the forms varies with cell type (1). In the mouse, in addition to the long form, three short forms have been described (2). The form encoded by clone PR-1, used herein, contains a region of 23 residues adjacent to the transmembrane domain, highly homologous to similar regions in receptors of other members of the cytokine receptor family as well as a proline-rich motif (3), which is essential for signal transduction. Both the long and the intermediate forms of the rat PRLR are able to induce expression of ß-lactoglobulin-chloramphenicol acetyl transferase or ß-casein-chloramphenicol acetyl transferase gene, whereas the short form of the receptor fails to do so (4). Similarly, the long and intermediate, but not the short, form of the rat PRLR were shown to induce expression of an early growth response gene (IRF-1) and mitogenesis in receptor-transfected lymphocytes (3). The coexpression in vitro of rat long and short forms of the PRLR resulted in a block of PRL signal to the milk protein gene promoter as a function of the concentration of this short form, suggesting that this form acted as a dominant negative in vitro (5, 6). Hence, it was concluded that the short form of the PRLR is not capable of transducing a lactogenic signal due to the truncation of a major portion of the cytoplasmic domain.
Moreover, PRL is known to be a mitogen (7) in addition to its function in promoting differentiation. Mammary gland development consists of multiple steps: establishment of the anlage during embryogenesis, ductal elongation and branching in puberty, and lobuloalveolar expansion in pregnancy (8). Recent studies using gene-targeting experiments have revealed some of the genetic components involved in each step of mammary gland development in mice (reviewed in Ref. 9). PTH-related peptide is required to establish the mammary gland anlage during development (10). The estrogen receptor is essential for ductal elongation (11), and genes required for lobuloalveolar development include those encoding PRL (12), PRLR (13), Janus kinase 2 (14), signal transducer and activator of transcription (Stat)5a and b (15, 16), progesterone receptor (17), cyclin D1 (18, 19), CCAAT enhancer binding protein (20), and A-myb (21), and very recently I
B kinase
(22). Although each of these genes has been shown to play an essential role in mammary gland development, their interrelationships are still largely unknown.
The use of the PRLR knockout model offers the possibility of directly examining the effects of PRLR-mediated signaling in vivo. Mice with only one copy of the PRLR gene (PRLR+/-) exhibit incomplete mammary gland development from midpregnancy and fail to lactate. It has been proposed that signaling using the amount of receptor produced by one copy of the gene is insufficient to activate the pathways responsible for functional mammary gland development (23).
The present study was carried out to elucidate the role of the short form of the PRLR in vivo. We developed a transgenic mouse model in which the full length cDNA for one of the short forms of the mouse PRLR (PR-1) (2) was expressed under the control of the polypeptide chain elongation factor 1
(eF1
) (24). Because this promoter is active in a wide range of mouse tissues, it should allow the expression of the transgene in all tissues during all stages of development. To elucidate the role of the PRLR short form, we expressed it in the PRLR+/- background. The resulting mice exhibited normal mammary ductal development and were able to lactate after their first pregnancy. These results strongly suggest that this short form of the PRLR is able to completely rescue mammopoiesis, in the PRLR+/- background.
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RESULTS
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Generation and Characterization of Transgenic Mouse Model
Transgenic mice expressing the PRLR short form called PR-1 (2) under the control of the eF1
promoter were generated by injecting one-cell 129 Sv PRLR+/- zygotes with the transgene construct. Before the injections, we tested the ability of the transgene construct to be expressed in cultured cells. Transfection of the plasmid containing the eF1
PR-1 construct into HEK-293 (human embryonic kidney cell line) cells resulted in the expression of the expected PRLR protein as revealed by chemical cross-linking experiments with [I125]PRL. Specificity of binding was determined using an excess of unlabeled ovine PRL (not shown). This in vitro study indicated that the intron splicing and eF1
promoter functioned appropriately in conjunction with the PR-1 cDNA.
Four transgenic founders, three females and one male, were identified by PCR using a pair of transgene-specific oligonucleotides. The integration status of the transgene was tested by Southern blot analysis. The transgene contains one BamHI site, and BamHI digestion of the genomic DNA from the transgenic mice released the predicted 5.4-kb transgene, after which the number of integrated copies was determined. Of the four founders, one female (N12) died prematurely, and one male (N30) was unable to transmit the transgene to its female offspring, probably as a result of the linkage to Y chromosome. The two other founders (N10 and N24) successfully transmitted the transgene in a Mendelian manner, thereby allowing the establishment of two independent transgenic mouse lines. The number of integrated copies was determined by Southern blot analysis using the specific probe (short form of PRLR) on genomic DNA coming from a PRLR-/- animal and corresponded to 6, 3, and 42 for N30, N10, and N24, respectively. Further studies were performed on offspring of N24, essentially based on the high number of copies of the transgene. The availability of wild-type or PRLR -/- fertile males allowed generation of all PRLR genotypes (+/+, +/-, and -/-) as transgenic or nontransgenic animals.
Analysis of Expression of the Transgene
mRNA expression of the PR-1 short form of the PRLR was analyzed in various tissues of N24 transgenic mice by Northern blot (Fig. 1
). To more accurately determine the expression level of the transgene and avoid the interference of the endogenous short form of PRLR, analyses were conducted using transgenic PRLR -/- females. All tissues examined were shown to express significant amounts of receptor short-form mRNA with the high relative levels in reproductive organs (essentially in mammary gland and uterus) as well as in lung and liver.

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Figure 1. Northern Blot Analysis of eF1- -PR-1 Expression in Tissues from a PRLR-/- Transgenic Female
Twenty micrograms of total cellular RNA from each of the indicated tissues from transgenic mouse were subjected to Northern blot analysis. The filters were hybridized with a 32P-labeled PR-1 cDNA probe. The filters were subsequently stripped and reprobed with the rpl7 cDNA as a loading control. Lanes 17 show transcripts from an adult female transgenic mouse corresponding, respectively, to ovary, mammary gland, uterus, kidney, muscle, lung, and liver.
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Long- and Short-Form Expression in Transgenic PRLR+/- Mice
We hypothesize that the failure of complete mammary development seen in the PRLR +/- is due to the loss of one copy of the gene. Therefore, we examined long and short forms of receptor expression and found that the long form was expressed uniformly in all transgenic and nontransgenic +/- mammary tissues, at approximately half the level of +/+ mammary tissues (Fig. 2
, A and B). As expected, the N24 transgenic mice expressed a high level of short receptor transcript (Fig. 2
, A and C).

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Figure 2. Expression of Relative Long and Short Form of PRLR in Mammary Gland from Wild-Type, Nontransgenic PRLR+/-, and Transgenic (N24) PRLR+/- Female Mice
A, Semiquantitative RT-PCR analysis of PRLR at d 18 of pregnancy. B and C, Quantification of long and short forms of mRNA levels from the above experiments normalized to glyceraldehyde-3-phosphate dehydrogenase levels. Each value is the mean ± SEM of six independent mice; *, P < 0.05.
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Rescue of Mammary Gland Development and Function in Transgenic PRLR+/- Mice
To evaluate the developmental effects of the PR-1 transgene, we examined mammary ductal outgrowth in N24, N10, +/-, and +/+ mice by analyzing whole-mount preparations of the fourth abdominal mammary tissues from d 14 of pregnancy to d 1 of lactation (Fig. 3
). Young virgin mammary tissues were also analyzed, and no modification between genotypes was noted (data not shown). In rodents, lobuloalveolar development during pregnancy is initially dependent upon increased PRL production by the pituitary, after which development becomes dependent upon production of placental lactogen. Morphology of the mammary tissue ductal network appeared similar in all genotypes of mice from puberty to the onset of pregnancy; however, alveolar structures that developed along the length of the mammary duct were strongly affected in +/- mice. In contrast, extended lobuloalveolar structures were abundant in transgenic mice of both the N10 and N24 lines, such that from midpregnancy, restoration of lobuloalveolar architecture was seen similar to that observed in wild-type mice. Moreover, the N24 line, which displayed a high copy number of the transgene, exhibited alveolar lumen with milk shortly after parturition.

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Figure 3. Whole Mounts of Mammary Gland from Wild-Type, Nontransgenic PRLR+/- Females and Transgenic PRLR+/- Mice
Whole mounts of four inguinal mammary glands from control (+/+) or heterozygous (+/-) nontransgenic or transgenic N10 and N24 females were prepared and stained with hematoxylin. Different stages of mammary development d 14 of pregnancy (d14) and the first (L1) day after birth were examined.
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Cell Proliferation in Transgenic Mammary Gland Development
To evaluate mammary epithelial cell proliferation at midpregnancy, we performed in vivo bromodeoxyuridine incorporation assay to determine whether the development of mammary ducts and alveoli resulted from an increase of cell proliferation. A large number of labeled cells was observed in transgenic PRLR+/- as compared with non transgenic PRLR+/- mammary tissue of mice (Fig. 4A
). Expression of the transgene clearly induced proliferative changes in the mammary tissue. Moreover, PRL may signal cells to grow and divide through a pathway involving MAPK. As shown in Fig. 4B
, only protein extracts from PRLR+/- N24 and wild-type mice demonstrated activation of MAPK accompanied by phosphorylation of the 42-kDa isoform of MAPK. This constitutes a confirmation of the observation already made by Das and Vonderhaar (25).

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Figure 4. Epithelial Cell Proliferation in Mammary Glands Tissues and MAPK Activity
A, BrdU was administered at d 10 of pregnancy and the mammary tissues were removed 2 h later. Paraffin sections of tissues were stained with anti-BrdU. Epithelial cell nuclei (300400) were examined per section of PRLR+/- (+/-) and PRLR+/- transgenic (+/- N24) mammary tissues. The values represent the average fraction of BrdU-positive epithelial cells per total number of epithelial cells of three different mice. *, P < 0.05. B, MAPK phosphorylation was measured by immunoblots using phophospecific p44/p42 MAPK antibodies; total cell lysates (40 µg) were loaded on a 10% SDS-polyacrylamide gel and analyzed by immunoblotting using antithreonine/tyrosine-phosphorylated, Erk1/2-specific antibodies. Membranes were stripped and reprobed using anti-Erk1/2 MAPK antibodies to check equal loading (lower panel). P-Erk1/2, Threonine/tyrosine-phosphorylated Erk1/2.
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To test directly the ability of the biological effect of the transgene, we performed transplantation experiment (transgenic +/- mammary tissue into non transgenic +/- host) with proper controls (Fig. 5
). All of the transplants had filled the pad fat, and morphological differences were observed in the transplants. We are now able to conclude that the observed rescue is autonomous to the mammary gland because alveolar development is seen in transplanted hosts at parturition.

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Figure 5. Whole-Mount Analysis of Epithelial Transplants at Day of Parturition
Transgenic PRLR+/- epithelium has filled the fat pad. Of the five separate transplants examined, all developed extensive lobuloalveoli. Transplanted mammary glands were stained with carmine alum on the day of delivery. Contralateral endogenous mammary epithelia of the PRLR+/- host exhibited structures severely underdeveloped.
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Mammopoiesis Is Rescued in Transgenic PRLR+/- Mice
To analyze the biological effect of PR-1 short form, young (6- to 10-wk-old) transgenic +/- females were bred with fertile males, and monitoring of pup development was performed to examine lactational ability. Surprisingly, the failure of lactation in nontransgenic mice was totally rescued in transgenic PRLR+/- mice. Pup weight was recorded, after litter size was normalized to eight pups. Lactational performance, as measured by pup weight, is represented in Fig. 6
. No difference was evident in terms of pup growth between litters from PRLR+/+ and transgenic PRLR+/- females, whereas PRLR+/- mice failed to support pup growth. Due to the possible PRL hypersecretion caused by the short form of PRLR, we measured PRL levels in wild-type, heterozygous transgenic and heterozygous nontransgenic females, at three stages of pregnancy (d 10.5, 14.5, and 16.5), and the results clearly indicate that there are no differences in PRL levels between any of the genotypes. Moreover, we examined mammary gland development in wild-type mice expressing the transgene. No phenotypic effects were seen in these animals during all stages of development of the mammary gland, and there was no alteration of pup weight.

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Figure 6. Average Growth Curves of Pups from Wild-Type and Transgenic Female Mice
Pup weight was measured each day until weaning. Litters were standardized to eight pups and mean of weight of each pup is shown from eight individual wild-type (triangles) and eight +/- transgenic (circles) mice. After calculation of regression curves, slopes are not significantly different.
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Milk Protein Gene Expression and Stat5 Activation in Rescued Transgenic PRLR+/- Mice
To determine the degree of differentiation of the mammary glands, we measured ß-casein and whey acidic protein (WAP) mRNA levels at d 18 of pregnancy and d 1 of lactation (Figs. 7
and 8
). Although both casein and WAP were abundant in control wild-type tissue at d 18, expression in five PRL +/- mice was reduced by approximately 90% (Fig. 7
, A and B, lane +/-). Although WAP and casein mRNA levels increased in heterozygous mice at parturition, they reached only 10% of the level seen in control mice (data not shown). In contrast, the transgenic PRLR+/- expressed ß- casein mRNA as well as protein (Fig. 7C
) at levels approximately equivalent to those of wild-type glands. WAP gene expression was significantly increased, but not to the level seen in +/+ glands. This demonstrated that the alveolar cells of transgenic animals achieved full differentiation. The presence and phosphorylation state of Stat5 were also analyzed in transgenic and nontransgenic mammary glands. We have shown that Stat5 phosphorylation in mammary tissue from late-pregnant PRLR+/- mice after the first pregnancy was very low (23), and at midpregnancy, the level of phosphorylation is very weak. In contrast, normal phosphorylation levels were observed in transgenic PRLR+/- derived from the N24 line that were able to lactate, demonstrating that the signaling pathway of PRL was activated (Fig. 8
).

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Figure 7. Expression of Steady-State Levels of ß-Casein and WAP mRNA and ß-Casein Protein in Mouse Mammary Glands
Mammary glands from wild-type (+/+), nontransgenic heterozygous (+/-), and heterozygous transgenic (N24) mice were removed at d 18 of pregnancy. Total RNA was isolated, and the expression of ß-casein and WAP was evaluated by RT-PCR. The quantification of casein (A) and WAP (B) expression was determined from the fourth inguinal gland. Values represent means ± SEM of five mice per group. Western blots were performed on 40 µg of total protein extracts from wild-type (+/+), heterozygous (+/-), and heterozygous transgenic (+/- N24) mice and revealed with polyclonal anti-ß-casein (C).
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Figure 8. Stat5a Activation in Mouse Mammary Gland
Cellular proteins from mammary tissue from wild-type (+/+), nontransgenic heterozygous (+/-), and heterozygous transgenic (+/- N24) 14 d pregnant mice were precipitated with anti-Stat5a antibodies, followed by Western blot analysis with antiphosphotyrosine (top) and anti-Stat5a (bottom).
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DISCUSSION
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Signaling through the PRLR is crucial for functional mammary gland development. Mice lacking both alleles of the PRLR gene are sterile; thus, the analysis of mammary development is difficult (13). We have shown that PRLR heterozygous mice exhibit a severe defect in lactation after the first pregnancy. The subsequent pregnancies result in lactational capacity sufficient for pup survival and normal growth rate but with a developmental lag of approximately 3 d due to slow onset of growth. Up to midpregnancy, PRLR heterozygous mice show normal ductal and alveolar development, but alveolar development stalled during late pregnancy, preventing successful lactation (26). This phenotype was also observed in mice deficient for the gene encoding galanin, a peptide controlling pituitary PRL release, and Stat5a, a member of the signaling pathway of the PRLR (15, 27). Transplantation experiments demonstrate that a direct action of lactogenic hormones to promote alveolar development is confined to the epithelium (26), suggesting that the defect is intrinsic to mammary epithelial cells and this hypothesis was confirmed by our recent studies (23).
After reintroducing multiple copies of the gene encoding a short form of mouse PRLR in PRLR +/- mice, mammary development and function were rescued. All of the earlier stages of mammary development in PRLR heterozygous mice were apparently normal, including the appearance of mammary epithelial buds, and a rudimentary ductal tree in the embryo, as well as branching morphogenesis, which takes place at puberty, and also the early stages of mammary development in pregnancy (28). Analysis of mammary development showed that before d 15 of pregnancy, ductal elongation, branching, and the number of lobules formed were similar between PRLR genotypes in response to the hormonal environment of pregnancy. Whereas impaired cellular proliferation of PRLR heterozygous mammary epithelia is evident in the late phases of pregnancy, we observed a clear increase in the proliferation rate in transgenic PRLR+/- mice and an extensive development of mammary architecture similar to the wild-type tissue. These results are in complete agreement with those of Das and Vonderhaar (25), who demonstrated, in NIH-3T3 cells stably expressing the same mouse PR-1 form of PRLR, that specific binding of PRL and mitogenic responsiveness were normal; these findings suggest that this short form of the receptor is able to transduce the mitogenic signal through similar pathways as the long form.
Although the PRLR+/- lobules were not expanded by milk secretion, suggesting a failure of the final stage of functional differentiation as the cause of failed lactation, the transgenic PRLR+/- lobules were able to synthesize the major components of the milk in abundance, as demonstrated by greatly increased synthesis of ß-casein and WAP. The analysis of milk composition in the animals showed no difference in terms of quality and quantity of proteins as compared with the wild-type control.
Alveolar cells of +/- females fail to reach full differentiation. We have demonstrated that a threshold level of Stat5 phosphorylation is required for the development of a functional mammary gland (23). In accordance with these effects of PRL, Stat5 phosphorylation in mammary tissue from PRLR+/- mice after the first pregnancy was very low (23), whereas normal phosphorylation levels were observed in transgenic animals in the +/- background.
A recent report (29) describes the novel observation that expression of the short form of the receptor is increased markedly in rat luteal cells, suggesting a role other than that of a dominant negative effect on Stat5 activation by PRL. The authors suggest that the short form may exert positive regulatory effects on Stat activation/turnover. Acquisition of Stat5b responsiveness to PRL is initiated after luteinization and is associated with increased PRLR expression, most strikingly an increase in the short form of the receptor, suggesting that the transition from granulosa to luteal cells is mediated either by a dominant negative effect of the short form of the PRLR on Stat5b activation or rather by a positive effect of the short form of the receptor.
In another study, expression of the rat F3 short form in lymphocytes failed to demonstrate any PRL-induced growth (3). Such an observation, which contradicts the results of the present study, may be explained by the cell type used. The sequence of the short form (mouse PR-1 or rat F3) may also be of major importance. Moreover, Wu et al. (30) reported in an abstract that treatment of HC11 mouse mammary cells with PRL resulted in increased ratios of short to long form receptor mRNA and different degrees of ß-casein gene expression. They suggest, in fact, that in this model the short form does not act as a dominant negative to expression of milk protein genes and supports the idea that an increase in the ratio short/long form could be responsible for ß-casein up-regulation.
In this study, we have confirmed our previous findings that PRLR+/- mice fail to undergo the final stages of mammary development required for successful lactation. We have taken advantage of this model to examine the role of the PR-1 short form of the mouse PRLR. Previous studies with the F3 short form of the rat PRLR demonstrated that alone it is unable to activate the PRL signaling pathway in vitro and in fact acts in vitro or in vivo as a dominant negative receptor, presumably by inducing inactive heterodimers with full-length PRLR or by competing for PRL binding with the long form (31). In contrast, the present studies demonstrate, for the first time, that in vivo a short form of the PRLR can act to augment both mitogenic responses and differentiation-dependent functions in the mammary gland. These results confirm and extend the data of Das and Vonderhaar (25), who demonstrated a mitogenic response in vitro to this same receptor short form.
Whether these apparently contradictory results are due to different intrinsic activities of the various short forms of the PRLR, or due to the different model systems used, it is clearly worthy of further investigation by testing the short forms function in homologous systems.
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MATERIALS AND METHODS
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Generation of eF1
-PRLR-PR-1 Transgenic Mice
The eF1
-PRLR-PR-1 transgene was constructed using the transgenesis vector p polyIII kindly provided by Dr. L. M. Houdebine (Institut National de la Recherche Agronomique, Jouy en Josas, France). The mouse PRLR PR-1 cDNA encoding amino acids 1303 kindly provided by Dr. D. Linzer (2) after EcoRI digestion was blunt-ended using Klenow DNA polymerase (Life Technologies, Inc., Gaithersburg, MD) and ligated into the SpeI site. Then it was inserted at the SpeI site downstream of the human eF1
promoter and part of SR
, a translation stimulator into the optimized vector pPolyIII, containing the terminal block of human GH, including the last intron and the polyadenylation site. The transgene was separated from plasmid vector sequences by NotI digestion and purified after agarose gel electrophoresis with Elutip-d columns (Schleicher \|[amp ]\| Schuell, Dassel, Germany) and ethanol precipitation. Microinjections into fertilized PRLR+/- oocytes derived from 129Sv pure background mice were performed. All mice were maintained in accordance with the institutional and European animal care policies.
Founders were identified by PCR analyses of tail DNA from 2-wk-old mice using oligonucleotides specific for the human GH terminator (sense primer: 5'-AAGTTCGACACAAACTCACA-3'; antisense primer: 5'-ACTGAGTGGACCCAACGCAT-3'); PCR conditions are available upon request. The number of integrated copies of transgene was determined by Southern blot analysis.
Northern Blot Analyses
Total RNA was isolated from different mouse tissues with Trizol (Life Technologies, Inc.) according to the manufacturers recommendations. Twenty micrograms of total RNA were separated by formaldehyde-agarose gel electrophoresis and transferred to nylon filters. Northern blot analysis was performed by standard techniques (32), using an
-32P- labeled fragment encompassing the coding region of the PR-1 gene as probe (Megaprime DNA labeling system; Amersham Pharmacia Biotech, Arlington Heights, IL) or ribosomal protein l7 (rpl7) as internal control.
Histology and Epithelial Transplantation
The fourth inguinal mammary glands from transgenic and control mice were removed and fixed in 4% formalin. Whole mounts were performed as described previously (33), using carmine alum staining. Formalin-fixed specimens were paraffin embedded and serially sectioned (5 µm).
Mammary tissues from no. 4 (inguinal) gland transgenic PRLR +/- were dissected out and transplanted into the no. 4 cleared fat pads of 3-wk-old recipient PRLR+/- hosts. Clearing of the endogenous mammary epithelium from the host animals involved the surgical excision of the no. 4 inguinal gland from the nipple to the lymph node. The excised portion of the gland was routinely whole mounted and stained using carmine alum to confirm that the epithelium was completely removed from the cleared fat pad. Eight weeks after transplantation, mice were bred and the transplants were removed at different stages of pregnancy, fixed, and stained.
Cell Proliferation
In vivo bromodeoxyuridine (BrdU) labeling was performed by ip injection of BrdU. Two hours before the mice were killed, BrdU (100 µg/g body weight) was injected ip on d 15 of pregnancy.
After embedding the mammary glands in paraffin and sectioning, sections were deparaffinized, hydrated, pretreated with 2 N HCl for 20 min at 37 C, and exposed to 0.01% trypsin at 37 C for 3 min. Incorporated BrdU was detected with anti-BrdU antibody conjugated to horseradish peroxidase (Roche Clinical Laboratories, Indianapolis, IN) according to the manufacturers instructions.
Semiquantitative RT-PCR Analysis
RNA was isolated from mammary gland at different stages of pregnancy. One microliter of each reverse transcriptase reaction (1/20 of total) was used in each 20 µl PCR primed with gene-specific oligonucleotides. Long and short forms of PRLR were detected using common 5'-GAGAAAA ACACCTATGAATGTC-3' (sense) and 5'-AGCAGTTCT TCA GACTTGCC-3'(antisense) or 5'-CCTTGAGACTAGA TTATTGGC-3'(antisense) primers, respectively, spanning multiple exons and giving two mRNA-derived 660-bp and 690-bp products.
Mouse casein and WAP mRNA expression was detected using 5'-ACTACATTTACTGTATCCTCTGAC-3' (sense) and 5'-GTGCTACTTGCTGCAGAAAGTACAG-3'(antisense) and 5'-TAGCAGCAGATTGAAAGCATTATG-3' (sense) and 5'-CACCGGTACCATGCGTTG-3' (antisense) primers, respectively. PCR products of 537 and 500 bp were amplified from reverse transcriptase reaction. Primers were designed according to the reported sequences in European Molecular Biology Laboratory/GenBank.
PCR was carried out for 15, 20, 23, and 30 cycles; the conditions were such that the amplification of the products was in the exponential phase. The PCR products were separated on a 1% agarose gel in TBE 1x (Tris-borate-EDTA, pH 8) stained with ethydium bromide, the intensity of signals were reported to the intensity of glyceraldehyde-3-phosphate dehydrogenase (internal control), and data were analyzed with a DC 120 digital camera (Eastman Kodak Co., Rochester, NY) coupled with Kodak 1D 2.0.2 software.
Immunoprecipitation and Western Blot
Total cell lysates (2 mg protein) were used for immunoprecipitation with the following antibodies: polyclonal anti-Stat5a (L20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) used at 1 µg/ml and antiphosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology, Inc., Lake Placid, NY). Immunoprecipitation and Western blotting were performed as described in Ref. 34 . Direct Western blots were performed on 40 µg of total protein extracts revealed with polyclonal anti-MAPK (ERK1/2) and monoclonal antiphospho-MAPK (ERK1/2) clone 12D4 (Upstate Biotechnology, Inc.) or polyclonal anti-ß-casein (kindly provided by D. Flint).
Statistics
Values were compared by Students t test, and P < 0.05 was considered statistically significant.
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ACKNOWLEDGMENTS
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We thank Dr. L.-M. Houdebine and Dr. D. H. Linzer for providing the transgenic vector pPolyIII and the PR-1 cDNA, respectively. We also gratefully acknowledge Dr. Hélène Buteau for transgene construction and Dr. David Flint for helpful discussions and critically reading the manuscript.
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FOOTNOTES
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This work was supported in part by grants from Institut National de la Santé et de la Recherche Médicale and Ministère de lEducation Nationale de la Recherche et de la Technologie (No. 1A010G).
Abbreviations: Brdu, Bromodeoxyuridine; eF1
, elongation factor-1
; PR-1, short form of mouse PRLR; PRL, prolactin; PRLR, PRL receptor; Stat, signal transducer and activator of transcription; WAP, whey acidic protein.
Received for publication May 17, 2002.
Accepted for publication February 28, 2003.
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