A Novel LacZ Reporter Mouse Reveals Complex Regulation of the Progesterone Receptor Promoter During Mammary Gland Development
Preeti M. Ismail,
Jie Li,
Francesco J. DeMayo,
Bert W. OMalley and
John P. Lydon
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498
Address all correspondence and requests for reprints to: John P. Lydon, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: jlydon{at}bcm.tmc.edu.
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ABSTRACT
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To further our understanding of progesterone (P) as an endocrine mammogen, a PRlacz knockin mouse was generated in which the endogenous progesterone receptor (PR) promoter directly regulated lacZ reporter expression. The PRlacz mouse revealed PR promoter activity was restricted to the epithelial compartment during the prenatal and postnatal stages of mammary gland development. At puberty, PR promoter activity was unexpectedly robust and restricted to the body cells within the terminal end buds and to the luminal epithelial cells in the subtending ducts. In the adult, the preferential localization of PRlacz positive cells to the distal regions of ductal side branches provided a cellular context to the recognized mandatory role of P in ductal side-branching, and segregation of these cells from cells that undergo proliferation supported an intraepithelial paracrine mode of action for P in branching morphogenesis. Toward the end of pregnancy, the PRlacz mouse disclosed a progressive attenuation in PR promoter activity, supporting the postulate that the preparturient removal of the proliferative signal of P is a prerequisite for the emergence of a functional lactating mammary gland. The data suggest that PR expression before pregnancy is to ensure the specification and spatial organization of ductal and alveolar progenitor cell lineages, whereas abrogation of PR expression before lactation is required to enable terminal differentiation of the mammary gland.
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INTRODUCTION
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MOST OF THE physiological effects of progesterone (P) are mediated by its intracellular receptor, the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors (1, 2). The PR is composed of two receptor isoforms: PR-A and -B, which have been shown to exhibit differential transactivational properties in vitro (3) and, more recently, in vivo (4). Characterization of the PR knockout (PRKO) mouse (5), in which both PR isoforms were simultaneously ablated by gene-targeting approaches, provided critical in vivo validation of this nuclear receptors indispensable role in the normal development and function of the ovary, uterus, brain, and mammary gland. Although nonessential for organismic survival per se, the spectrum of phenotypes exhibited by the PRKO female reiterated the functional versatility of the PR in ensuring full reproductive capacity and the perpetuation of the species.
Among the wide range of PRKO reproductive phenotypes examined, the PRKO mammary defect, consisting of an impairment in pregnancy-associated tertiary ductal side-branching and alveologenesis, furnished much needed clarification with respect to the proliferative role of P in this tissue (6). Noteworthy was the observation that removal of the mammotrophic effects of P resulted in a marked reduction in the susceptibility of the PRKO mouse to carcinogen-induced mammary tumorigenesis (7), underscoring the complicity of this hormonal signaling pathway in facilitating the genesis and/or progression of this mammary tumor type. This finding also concurred with the conclusions of a number of recent clinical studies (8, 9, 10), which reported that combined estrogen (E)-P postmenopausal hormone replacement therapies increased breast cancer risk beyond that observed for E treatment alone.
Considering the pivotal role of P in enabling mammary morphogenesis to proceed through a developmental cycle that incorporates the stages of pregnancy, lactation, and involution (11), and because completion of this cycle early or late in reproductive life can attenuate or accentuate breast cancer risk, respectively, reviewed in Ref. 6 , disclosing the mammary cell lineage(s) in which the PR is expressedand how this expression is hormonally controlled in vivois an imperative.
Toward this goal, the lacZ reporter encoding ß-galactosidase (ß-gal) was knocked into exon 1 of the murine PR gene by gene-targeting approaches to accurately chart the endogenous pattern of PR promoter action in a spatiotemporal context. The ability of this new PR reporter mouse, the PRlacz mouse, to accurately monitor the spatiotemporal regulation of PR promoter activity in the pituitary gland, uterus, and ovary, three classic progestin target tissues, validated the PRlacz mouse as a legitimate research tool with which to explore the regional, temporal, and hormonal regulation of the PR promoter in the less well characterized, but equally important, P-responsive target tissuethe mammary gland. In addition to recent transgenic models (12, 13), the PRlacz mouse represents an ideal reporter with which to confirm or explore novel E- and/or P-signaling pathways in situ and portends the expansion of a new subfamily of mouse models in which heterologous genes are expressed under the governance of the PR promoter in vivo.
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RESULTS
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Targeted Insertion of the LacZ Reporter into the Murine PR Gene
Figure 1A
diagrams the knockin strategy to target the insertion of the lacZ reporter into exon 1 of the murine PR gene by homologous recombination. In the design of the targeting vector (Materials and Methods), disruption of presumptive estrogen response elements was avoided (14, 15). Containing its own start codon and nuclear localization signal, the lacZ reporter was inserted 120 amino acids (aa) downstream of (and in-frame to) the initiating methionine for the PR-B isoform (16); a short region (122 aa) of the N-terminal domain, containing the initiating methionine for the PR-A form, was deleted with this knockin strategy. The positive/negative selection approach was used to enrich for targeted events in embryonic stem (ES) cells (17); by Southern analysis, a targeting frequency of approximately 10% was achieved. Using two independent targeted ES cell lines, panel B shows a confirmatory Southern of resultant wild-type, PRlacZ, and PRKOlacZ mouse genotypes derived from one of these cell lines; as for the 5' probe (shown in panel B), the 3' probe did not reveal aberrant integration events in the clones analyzed (data not shown). The PRlacZ mouse, heterozygous for the lacZ insertion, is a phenocopy of the wild type, whereas the PRKOlacZ mouse, homozygous for the lacZ insertion, is a phenocopy of the previously described PRKO mouse (5); for much of the studies described herein, we have focused our analysis on the PRlacZ mouse.

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Figure 1. Insertion of the LacZ Gene into Exon 1 of the Murine PR
Panel A outlines the gene-targeting strategy to knock-in the LacZ gene into exon 1 of the mouse PR gene. The in-frame insertion of the LacZ gene positioned this reporter cassette 120 aa downstream of the initiating methionine for PR-B, with attendant removal of the initiating methionine for PR-A; pertinent restriction sites are shown. Panel B shows a Southern genotypic analysis of wild-type, PRlacZ, and PRKOlacz mice, using the 5' probe as indicated in A. In panel C, double immunofluorescence demonstrates that the LacZ gene [LACZ; shown as green (FITC detection)] and PR gene [PR; shown as red (TRITC detection)] are expressed in identical luminal epithelial cells (MERGED; shown as yellow) in the mammary gland of an adult (12 wk old) PRlacz female; scale bar denotes 10 µm. The white arrowhead indicates a cell coexpressing LacZ and PR, and the yellow arrowhead shows a cell scoring negative for LacZ and PR expression.
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Double immunofluorescence was employed to verify that the PRlacz mouse exhibits cellular coexpression for lacZ and PR. As shown for the mammary gland (Fig. 1C
), primary antibodies to ß-gal (ß-gal encoded by LacZ) and to PR (as visualized by fluorescein isothiocyanate (FITC) (green) and tetramethlyrhodamine isothiocyanate (TRITC) (red) fluorescence, respectively) clearly showed that LacZ expression colocalized faithfully with endogenous PR expression.
Before determining PR promoter regulation in the mammary gland, the PRlacz mouse was tested for its ability to accurately detect PR promoter activity in situ in three previously characterized P-target tissues: the pituitary gland, uterus, and ovary.
The PRlacz Mouse Faithfully Detects PR Promoter Activity in the Pituitary Gland and Uterus
The endocrine phenotypes of the PRKO female highlighted the functional importance of the P-signal in the elicitation of endogenous and E-induced preovulatory gonadotropin surges and underscored the functional importance of pituitary derived (as well as hypothalamic derived) PR-mediated signaling in the normal progression of the ovarian cycle (18, 19, 20). In Fig. 2A
, the PRlacz mouse demonstrated that the PR promoter was not induced in the pituitary gland of the prepubescent PRlacz female (3 wk old). However, pituitary whole mount (and sections thereof) revealed significant PR promoter activity in the anterior lobe of the pituitary gland of the sexually mature (16 wk old) female (Fig. 2B
); PRlacz was not detected in the neural lobe. As expected, administration of P suppressed PRlacZ expression in the adult anterior pituitary gland (compare panel C with B). P-induced down-regulation of PRlacz expression was via the PR as evidenced by the inability of P to down-regulate PRlacZ expression in the PRKOlacZ pituitary (panel D). Interestingly, abrogation of PR function in the PRKOlacz mouse did not preclude development of this pituitary cell lineage. Based on previous reports on the primate and rat (21, 22), the primary secretagogue expressing the PR in the anterior pituitary gland is the gonadotrope; however, recent in vitro investigations suggest that lactotropes also express the PR in the murine anterior pituitary gland (23)a clear distinction from the rat and primate (24).

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Figure 2. Modulation of PR Promoter Activity by E and P in the Anterior Pituitary Gland, Uterus, and Uterine Vasculature
Panels AH show pituitary whole mounts and associated sections; black arrows indicate PRlacz positive cells in the anterior lobe (AL); in panel B, NL indicates neural lobe. Inset in panel G indicates anterior pituitary cells positive for ER- expression (arrow). Scale bars in panel A are 1 mm and 50 µm for whole mount and corresponding sections, respectively, and apply to panels AH, whereas the scale bar in inset in panel G denotes 25 µm. Panels IK show a segment of lacZ stained uterine whole mounts with attendant transverse sections from untreated, P-, and E-treated ovariectomized PRlacz mice, respectively. In the section shown in panel K: S, LE, GE, and M indicate stroma, luminal epithelium, glandular epithelium, and myometrium, respectively; scale bar in panel I (whole mount) represents 1 mm and applies to all uterine whole mounts shown, whereas scale bar in panels I and K (sections) denotes 200 µm. Panels L and M show whole mounts of uterine blood vessels (arrow) from untreated and E-treated ovariectomized PRlacz mouse, respectively; note the E induction of PR expression in panel M. Panels N and O reveal that PR expression is localized to a smooth muscle cell (SMC) but is not expressed in the endothelial cell (E); scale bar in panel L represents 200 µm and also applies to panel M; scale bars in panels N and O correspond to 50 and 20 µm, respectively.
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Ovariectomy resulted in a significant reduction in PRlacZ expression in the pituitary of the adult female (panel E) as compared with the intact female (panel B), whereas E administration (panel F) reinstated the regional distribution and level of PRlacz expression. This result not only underscored the importance of ovarian-derived E in the induction of the PR in the anterior pituitary gland but also demonstrated the fidelity of the PRlacz mouse in tracking P and E modulation of PR promoter activity in vivo. Despite estrogen receptor-
(ER-
) expression in the pituitary of the adult male (25) [panel G, inset (black arrows)], PRlacz expression was not detected; however, PRlacz was induced with exogenous E to levels comparable to those detected in the pituitary of the similarly treated ovariectomized female (panel H). Although the E induction of PRlacz expression in the male pituitary is of uncertain physiological relevance, this observation does serve to showcase the potential utility of the PRlacz mouse as a powerful mouse platform with which to uncover hitherto unsuspected E- and P-signaling pathways in vivo.
Historically, the uterus has represented the archetypal target-tissue for P action; like the pituitary, this tissue has been studied extensively with respect to steroid hormone modulation of PR and ER-
expression (26, 27). Panels IK show the effect of P or E administration on PRlacZ expression in the epithelial, stromal, and myometrial cellular compartments of the uterus. Significant PRlacZ expression was observed in the luminal and glandular epithelial compartments within the uterus of the untreated ovariectomized mouse (panel I); however, after P administration, this expression was not detectable in all uterine cell types (panel J). In concert with a classic E-induced uterotropic response, E administration resulted in a significant increase in the level of PRlacz expression (panel K). In agreement with recent findings by our group and others (26, 27), E treatment for 4 d resulted in a down-regulation of PRlacz expression in the hyperplastic luminal epithelial cell layer, whereas PRlacZ expression was significantly increased in the glandular and myometrial compartments; PRlacZ expression was also increased in the stromal compartment.
Although PRlacz expression was not detected in the uterine vasculature of the ovariectomized PRlacz mouse (panel L; arrow), PRlacZ expression was clearly observed after E treatment (panel M; arrow) and was restricted to the outer smooth muscle cell layer of the capillary (panels N and O); PRlacz expression was also detected in the uterine vasculature of the intact virgin (data not shown).
PRlacz Expression Is Acutely Induced in Granulosa Cells of the Preovulatory Follicle
Unlike in the pituitary and uterus, intraovarian PR expression is transiently induced in granulosa cells of the preovulatory follicle by LH (reviewed in Ref. 28). Because of the distinct regulation of the PR promoter in this tissue, the ovary was included to determine whether this alternative mode of PR promoter regulation could also be shown in the PRlacz mouse. Due to the low level of LH-induced PRs in this tissue (29), the PRKOlacZ mouse was employed for more sensitive detection (both PR alleles harbor the lacZ knockin mutation in the PRKOlacz mouse); despite a defect in follicular rupture (5), the PRKO ovary develops normally throughout the preovulatory stages and undergoes luteinization.
PRlacZ expression was not detected in the ovary of the 21-d-old mouse without hormone treatment (data not shown) nor 48 h after the administration of pregnant mare serum gonadotropin (PMSG) (Fig. 3A
); PMSG is an analog of FSH. However, as early as 4 h after human chorionic gonadotropin (hCG) treatment, PRlacZ expression was clearly observed in the granulosa cells of preovulatory follicles (panel B) with highest levels of PRlacz expression attained within 8 h of hCG administration (panel C); hCG is equivalent in action to LH. After ovulation (1416 h after hCG treatment in the mouse) and subsequent luteinization, PRlacZ expression was undetectable in resultant corpora lutea, 24 h after hCG treatment (panel D). Panel E shows a higher magnification of a preovulatory follicle, notice PRlacz expression is restricted to the mural granulosa cells (panel E, blue arrowhead) but is not expressed in the oocyte (panel E, arrow) or the cumulus cells (panel E, black arrowhead), supporting PRs dedicated role in follicular rupture but not necessarily in oocyte viability after ovulation (30). In the case of the oviduct, PRlacz expression was predominantly localized to the tall columnar epithelial cells (ciliated and nonciliated) that line the oviductal lumen (panel F, gray arrowhead); a small number of smooth muscle cells dispersed within the oviductal wall also expressed PRlacz (panel F, brown arrowhead).

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Figure 3. Induction of PRlacZ Expression in Granulosa Cells of the Preovulatory Follicle
After 48 h of PMSG administration, panels AD show lacZ stained PRKOlacZ ovarian whole mounts and sections from untreated and from 4, 8, and 24 h after hCG treatment, respectively. Scale bar in panel A (whole mount) corresponds to 1 mm and applies to all ovarian whole mounts; scale bar in panels A and B (sections) represents 500 µm, scale bar in panel B (section) applies to sections shown in panels C and D (CL in panel D denotes corpus luteum). Panel E shows a higher magnification of a preovulatory follicle; notice PRlacz expression in the mural (blue arrowhead) but not in the cumulus (black arrowhead) granulosa cells or in the oocyte (O). Panel F reveals that the majority of PRlacz expression in the oviduct is confined to tall columnar epithelial cells that line the oviduct (gray arrowhead), with a small number of smooth muscle cells scoring positive for PRlacz expression (black arrowhead); scale bars in panels E and F are 200 µm.
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In sum, the spatiotemporal expression profile for PRlacz in the pituitary, uterus, and ovary (three distinct target tissues for P action) endorse the PRlacz mouse as a valid model system with which to further examine PR promoter function in less well-characterized P-target tissues, such as the mammary gland.
Distinct Differences in the Level and Spatial Distribution of PRlacZ Expression in the Mammary Gland as the Mouse Develops from Puberty to Sexual Maturity
Whole mount analyses revealed robust PRlacZ expression throughout the epithelial structures within the mammary gland of the peripubertal (5 wk old) mouse (Fig. 4A
); a similar expression pattern was observed in the neonatal anlagen (data not shown). A conspicuous high level of PRlacZ expression was observed in the multicell-layered terminal end buds (TEBs), transverse and longitudinal sections of which (panels B and C, respectively) demonstrated that PRlacZ expression was exclusively localized to the body cells as well as to the monolayer of luminal epithelial cells that lines the subtending duct; PRlacZ expression was not detected in the cap and myoepithelial cells, periductal fibroblasts, stromal adipocytes, or in the vasculature. After the first number of estrous cycles, the mammary gland of the 8-wk-old or juvenile was shown to exhibit the highest level of PRlacZ expression as compared with all other stages of mammary development; furthermore, most luminal epithelial cells scored positive for PRlacZ activity (panels DF). Remarkably, in the sexually mature adult mouse (16 wk old) (panels GI), the level of PRlacZ expression was significantly attenuated and adopted a nonuniform expression pattern as compared with the juvenile female (compare panel I with F).

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Figure 4. Dynamic Changes in the Level and Spatial Organization for PRlacZ Expression in the Mammary Gland of the Nulliparous Virgin Mouse
Panel A shows PRlacz expression in the whole mount mammary gland of the 5-wk-old female; scale bar denotes 0.5 cm and also applies to panels D and G; the lymph node (LN)proximal to the nipple as a referenceand TEB are indicated. Panels B and C represent transverse and longitudinal TEB sections, respectively; 5-bromo-2-deoxyuridine-positive cells are indicated by arrowheads in panel C; scale bar in panels B and C equals 0.1 mm. Low and high magnification of mammary whole mount PRlacZ expression in the 8-wk-old virgin is indicated in panels D and E; inset in D shows immunohistochemistry for PR in the TEB, supporting our observations in the PRlacZ mouse. Panel F reveals the high level of PRlacZ expression at this stage of mammary development; notice that most of the luminal epithelial cells are expressing PRlacZ (scale bars in panels E and F are 0.5 and 0.3 mm, respectively). A significant decrease in the level of PRlacz expression in the sexually mature adult virgin is clearly discernible in panels GI; high magnification of medial ducts (and sections thereof) show a nonuniform expression pattern for PRlacz expression. Scale bars in panels H and I represent 0.3 mm and 0.1 mm, respectively.
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PR Promoter Activity Is Exclusively Associated with a Specific Cell Type in the Mammary Gland
Figure 5
shows a high magnification of mammary sections from the PRlacz juvenile (A), the PRKOlacz adult (B), and the PRlacz adult (C). Based on morphology and ability to express PRlacz, two luminal epithelial cell types were clearly discernible in the mammary gland of the juvenile (A). The first cell type contained large elliptical nuclei and exhibited robust PR promoter activity (blue arrow), whereas the second cell type, which was PRlacz negative, harbored an elongated nucleus and protruded into the ductal lumen (red arrow); in most cases, one to three PRlacz negative cells were interposed between two PRlacz positive cells. Interestingly, examination of the adult PRKOlacz mammary gland (B) revealed a similar PRlacz expression pattern, supporting the postulate that a functional P-signaling pathway is not essential for the de novo development of cells that normally express PR in the juvenile, nor is it required for the establishment of the uniform expression pattern, but it is required for the elaboration of the nonuniform pattern observed in the adult PRlacz mouse (C).

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Figure 5. PR Promoter Activity Is Associated with a Distinct Morphological Cell Type in the Mammary Gland
A, A section through a duct within the mammary gland of the juvenile mouse. Notice PRlacz expression is restricted to luminal epithelial cells with large oval nuclei (blue arrow), whereas smaller cells with elongated nuclei score negative for PRlacz expression (red arrow). Panel B demonstrates that in the adult PRKOlacz mammary gland, the spatial cellular distribution pattern for PRlacz expression is maintained. Panel C shows the nonuniform expression pattern for PRlacz expression in the mammary gland of the adult PRlacz mouse. In contrast to A and B above, the majority of PR negative cells (black arrow) exhibit a similar morphology to PRlacz positive cells. Scale bar in A is 20 µm and also applies to B; scale bar in C denotes 20 µm.
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Unlike in the PRlacz juvenile and adult PRKOlacz mammary gland, the PRlacz negative cell population (black arrows) in the mammary gland of the PRlacz mouse appeared morphologically similar to the PRlacz positive cells (blue arrows).
The PR Promoter Is Distinctly Modulated by E and P in the Mammary Luminal Epithelial Cell
Because the establishment of a uniform cellular pattern for PRlacZ expression in the murine mammary gland coincided with the onset of puberty and because the transition of this expression pattern to a nonuniform pattern in the sexually mature adult correlated with the cumulative exposure of the gland to cyclical elevations of E and P during the estrous cycle, the ovariectomized PRlacZ mouse (16 wk old) was treated with E, P, or E + P to evaluate the potential of these hormones to modulate the level and spatial pattern of PRlacZ expression in the luminal epithelial compartment as previously observed in Fig. 4
. As shown in Fig. 6
(AC), 2 wk after ovariectomy, PRlacZ expression was markedly abrogated in the mammary gland of the adult female (compare Fig. 6
, AC with Fig. 4
, GI). Administration of E alone (panels DF) significantly induced PRlacZ expression to a level that was observed in the intact 8-wk-old juvenile female (Fig. 4
, DF); note also the establishment of a uniform pattern for PRlacZ expression in response to E (Fig. 6F
). Panels GI demonstrate that P treatment alone does not alter the PRlacZ expression pattern previously observed in the untreated ovariectomized female; compare panels GI with AC. However, P, in combination with E was sufficient to reestablish the nonuniform expression pattern for PRlacZ activity normally observed in the adult gland; note the increased tertiary ductal side-branching and associated alveologenesis in response to the inclusion of P; compare panels JL with DF.

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Figure 6. E and P Modulation of PRlacZ Expression in the Mammary Gland
A and B, Low and high magnification of whole mounts from a 16-wk-old untreated ovariectomized PRlacZ mouse, respectively; a section of a medial duct reveals negligible PRlacz expression (panel C). After E administration, robust PRlacZ expression is observed in the mammary gland of the ovariectomized PRlacz mouse (panels D and E). Apart from an increase in PR promoter activity per cell; almost every luminal epithelial cell expresses PRlacz with this hormone treatment (panel F). Similar to panels AC, P administration has no effect on PRlacz expression in the mammary gland of the ovariectomized PRlacz mouse (panels GI). However, P in combination with E results in extensive ductal branching and alveologenesis (panels J and K); note the nonuniform pattern for PRlacz expression in the main ducts (panel L) and the concentration of cells expressing PRlacz in the newly formed ductal side branches (panel L, inset). Scale bars in panels A and D represent 2 mm; scale bar in panel D applies to whole mounts shown in panels G and J. Scale bar in panel B denotes 0.2 mm and also applies to panels EK, whereas scale bar in panel C equates to 0.1 mm and applies to panels FL.
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PRlacZ Expression in the Embryonic and Male Mammary Gland
Motivated in large part by the significant PRlacZ expression observed in the peripubertal mouse (and neonate; data not shown) as shown in Fig. 6
, we examined whether PRlacZ expression existed in the embryonic mammary gland (Fig. 7
, AC). Unanticipated was the finding that PRlacZ expression was clearly discernable in the primary lactiferous duct and associated primordial ductal branches within the 16-d-old female embryonic mammary gland (panels A and B; black arrows); note also PRlacZ expression in the submandibular salivary glands (31) (panel A; blue arrow). A further surprise was the detection of PRlacZ expression in the mammary rudiment of the 14-d-old male embryo (panel C; black arrow) and that this expression was also observed in the vestigial epithelial duct of the 16-wk-old adult male mammary gland [panels D (black arrow) and E]. Unlike most mouse strains in which the male mammary epithelial remnant degenerates during embryogenesis (32, 33), our 129SvEvXC57BL/6 sub-strain joins a small subgroup of mouse strains (34, 35) in which the epithelial remnant in the male gland is not completely destroyed in response to fetal androgens. Moreover, in response to E (panels FH) or E plus P (panels I and L), this primitive mammary epithelial structure exhibited limited progression from the nipple region to the lymph node; interestingly, in the case of the E- and P-treated gland, limited ductal side branching was observed concomitantly with the presence of TEB-like structures located at the advancing epithelial front (panel K). Again, as with the female, E induced a uniform cellular pattern for PRlacZ expression (panel H), whereas the coadministration of P resulted in a nonuniform cellular organization for PRlacZ expression (panel L); panel K represents a cross-section through a typical TEB-like structure in response to E and P.

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Figure 7. PR Promoter Activity in the Embryonic and Male Mammary Gland
Panels A and B show low and high magnification of a mammary whole mount from a 14-d-old female embryo (black arrow); blue arrow in panel A denotes PRlacz expression in the submandibular salivary glands. Panel C reveals PRlacz expression in a section of the male embryonic mammary gland (arrow); E represents the embryonic epidermis. PRlacz expression is clearly evident in the rudimentary ductal structure in the adult male mammary gland (panel D) (arrow) and higher magnification (panel E). Panels FG show the response of the male mammary gland to E treatment; note a uniform pattern for PRlacz expression in the main ducts (panel H). In the case of E and P treatment (panels IL), limited ductal extension, side-branching, and TEB formation is evident; panels K and L show PRlacz expression in a TEB-like structure and a nonuniform expression pattern for PRlacz in the main ducts. Scale bar in panel C, 50 µm. Scale bars in panels D, E, G, H, I, and J represent 0.5 cm, 0.3 mm, 0.2, 0.1 mm, and 0.5 mm, respectively; scale bar in panel D also applies to panel F, whereas scale bar in panel H also applies to panels K and L.
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Progressive Attenuation of PRlacZ Expression as the Mammary Gland Attains Functional Differentiation
Figure 8
profiles PRlacZ expression in the inguinal mammary gland during early pregnancy (d 4) (panels AD); late pregnancy (d 18) (panels EG); the day of parturition (panel H); lactation (d 7) (panels IL); and involution (d 7) (panels MP). A noticeable feature of PRlacZ expression during early pregnancy was its predominant localization to the distal tips of newly formed tertiary side branches (panels B and C; black arrows). During this phase of the mammary cycle, the mammary gland undergoes significant epithelial proliferation; note in panel D, a clear demarcation between those cells that express PRlacZ (blue arrowhead) and those undergoing proliferation (brown arrowhead), supporting the involvement of a complex paracrine circuitry in the mediation of the P-induced proliferative signal during this stage of mammary development. As mammary gland development progressed toward the end of pregnancy and terminal differentiation, the level of PRlacZ expression was significantly reduced, with the majority of PRlacZ positive cells localized to the main ducts (panels F and G; black arrow); at the onset of parturition, PRlacZ expression was not detected (panel H). In the case of the lactating mammary gland, PRlacZ expression was negligible but could be induced by E; as recently shown for the rat (36), the inset in panel K (white arrow) revealed a significant level of ER-
expression during this phase of mammary development (ER-ß expression was not evaluated in this study). During involution, PRlacZ expression was not detected (panels MO); however, as for the lactating gland, PRlacZ expression was induced by E during this stage of mammary development (panel P).

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Figure 8. Marked Changes in PR Promoter Activity as the Mammary Gland Attains Functional Differentiation
A, Mammary gland whole mount from a 4-d pregnant PRlacz mouse; note the localized expression for PRlacz to the distal tips of newly formed ductal side branches (panel B; blue arrows). Panel C shows a section through one of these typical side-branches; a cluster of cells expressing PRlacz is clearly visible (panel C and inset). Panel D reveals a clear segregation between PRlacZ positive cells (blue arrow) and cells undergoing proliferation (brown arrow). Panels EG show a significant attenuation in the level PRlacz expression in the gland as the mouse progresses toward late pregnancy (18 d); on the day of birth, PRlacz expression was not evident (panel H). Few cells expressed PRlacz in the lactating mammary gland (panels IK); however, PRlacZ expression was induced with exogenous E panel L and the inset in panel K shows the lactating gland expresses ER- (white arrow). Although PRlacz expression was not detected in the whole mounts (panels M and N) or sections (panel O) of mammary glands undergoing involution, PRlacz expression could be induced by administered E (panel P). Scale bars in panels A, E, I, and M represent 0.5 cm; scale bars in panels B, F, G, JL, and NP denote 0.2 mm; scale bar in panel C (including inset) equates to 50 µm, whereas scale bars in panels D and H correspond to 20 µm and 0.5 mm, respectively.
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Localized PRlacZ Expression to Newly Formed Ductal Side Branches in Response to P
The significant concentration of cells expressing PRlacZ toward the tip of the extending tertiary side branch during early pregnancy (Fig. 8B
) coupled with our previous observation that P can modulate the cellular pattern for PRlacZ expression as the mammary gland progresses from the pubescent to the adult stages of development (Fig. 4
), prompted us to evaluate whether the administration of P alone to the intact adult could recapitulate the branching pattern and cellular organization for PRlacZ expression that occurs during early pregnancy. Figure 9
shows that the administration of P to intact adult females (12 wk old) resulted in an increase in tertiary ductal side branching, but unlike E plus P treatment, did not elicit alveologenesis (panels AD). Its important to note that to avoid the presence of a significant number of ductal side-branches before hormone administration, the 12-wk-old was used, rather than the 16-wk-old-nulliparous mouse. Importantly, although P caused a down-regulation of PRlacZ expression in the main ducts, as observed during early pregnancy; a significant concentration of cells expressing PRlacZ was observed toward the distal portion of P-induced side-branches (Fig. 9
, E and F).

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Figure 9. Down-Regulation of PR Promoter Activity by P in the Main Ducts of the Mammary Gland But Not in Newly Formed Side Branches
Panels A and B show mammary gland whole mounts obtained from an intact untreated and P treated-PRlacz virgin (12 wk old); note the overall decrease in PRlacz expression and increased branching in panel B. Compared with untreated virgin (panel C), panels DF reveal a selective suppression by P of PRlacz expression in the main ducts (black arrows) and a contemporaneous increase in the number of P-induced ductal side branches (blue arrows), which contain high concentrations of cells that express PRlacZ at their growth points. Scale bars in panel A and C denote 0.5 cm and 0.1 mm and also apply to panels B and D, respectively. Scale bars in panel E and F represent 0.2 mm and 50 µm, respectively.
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DISCUSSION
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Although the PRKO mouse underscored the functional importance of the PR to female fertility (5), progress in understanding the underlying cellular mechanism(s) by which this transcription factor exerts its physiological effects has been hampered by insufficient information concerning the cell lineages in which the PR is expressed and how this expression is hormonally controlled in situ.
To address this issue, we employed experimental mouse genetics and the lacZ reporter to define the PRs promoter activity in its appropriate physiological context, at both the whole mount and histological level. Rather than using a conventional transgenic approach, the effectiveness of which is dependent upon the inclusion of all regulatory elements of the gene in question and on the site of integration, we adopted a knockin strategy to target the lacZ reporter to the authentic PR locus.
Our initial studies on the pituitary, uterus, and ovary demonstrated the utility of the PRlacZ mouse to unequivocally identify those cell types that express this transcription factor; furthermore, the PRlacZ mouse detected the well recognized induction of PR promoter activity by E and LH as well as its repression by P.
For the majority of the studies presented herein, we have focused on applying the PRlacz mouse to defining the spatiotemporal activity profile of the PR promoter during the prenatal and postnatal stages of mammogenesis, including lactation and involution. In agreement with previous studies (37, 38, 39), which used molecular and immunoprobes to PR, the PRlacz mouse demonstrated that PR promoter action is restricted to the epithelial compartment of the mammary gland and specifically to the luminal epithelial cell lineage. Remarkably, although the PRKO mammary phenotype is not manifest until adulthood, the highest levels of PR promoter activity were observed during the embryonic, neonatal, and juvenile stages of mammary gland development. Although the functional significance of this early promoter activity is unknown, this observation supports the emerging hypothesis, shared by our group and others (37, 39), that early expression of the PR may serve to irrevocably commit a cell toward an alveolar developmental pathway later in mammary development. The existence of PRs in the embryonic male mammary gland would suggest that this developmental process is not selectively imprinted in the female; however, due to the absence of ovarian hormones, this process is not realized in the adult male.
Although the analysis of the PRlacz mammary gland confirmed the exclusivity of the luminal epithelial cell with respect to PR expression, this expression is not static but undergoes marked spatiotemporal changes: first, during the transition from the juvenile to the nulliparous sexually mature adult; and second, during the mammary cycle of pregnancy, lactation, and involution.
Consistent with recent immunofluorescent studies (39), the PRlacz mouse demonstrated that the cellular distribution for PR promoter action in the mammary gland underwent a striking change from a uniform in the juvenile to a nonuniform pattern in the nulliparous mature adult. Steroid-treated ovariectomized PRlacz mice underscored the importance of ovarian E as the primary hormonal cue for the establishment of the above uniform expression pattern in the juvenile mammary gland, whereas ovarian P was required to generate the nonuniform pattern for PRlacz expression in the gland of the young adult. Because neither apoptosis nor proliferation are prominent features during this stage of mammogenesis (40), it is more likely that cell-selective down-regulation of the PR promoter is responsible for the establishment of the nonuniform expression pattern for PR. The presence of a nonuniform expression pattern for PR in both the adult human (41) and rodent (39, 42) mammary gland suggests an evolutionary conserved role for this cellular organization in P-induced mammary morphogenesis; the conspicuous absence of this cellular organization in the recently reported CCAAT/enhancer binding protein ß knockout mammary gland (39), in which ovarian steroids fail to elicit epithelial proliferation, would further support this proposal.
During early pregnancy, PRlacz expression was evident throughout the mammary ductal architecture with prominent expression at the distal regions of advancing tertiary side branches. This observation is congruent with recent immunohistochemical studies (43) and supports the notion that the regional concentration of PR positive cells at the growth points of these ducts is particularly important for their extension into the mammary fat pad. As pregnancy progressed toward parturition, the PRlacz mouse clearly showed that PR promoter activity attenuated in the mammary gland, with minimal activity during lactation and involution. Interestingly, administered E could induce a nonuniform PRlacz expression in the periparturient mammary gland, suggesting that PR positive cells in the mammary gland of the prepregnant animal may still exist during this stage of mammary gland development.
Application of the PRlacz mouse to profile PR promoter activity during mammary gland development has highlighted established as well as relatively new questions regarding the role of P as an endocrine mammogen. These questions include: 1) Does early PR expression determine mammary cell fate? 2) Are the dynamic changes in the PRs spatiotemporal expression pattern essential for normal mammary morphogenesis? and 3) What regions of the PR promoter are required for its hormonal regulation during mammary gland development? Possible future experiments to address the first question include the use of the PRlacz mouse in combination with cell-sorting and transplantation approaches to determine whether isolated PR positive cells can generate ductal, alveolar, or both mammary epithelial structures. Our ongoing studies to address the second question include conditional transgenic approaches to spatiotemporally control PR expression in the PRKO mammary gland, whereas the recent isolation of bacterial artificial chromosomes containing the entire PR promoter (44) promise to offer significant advances toward addressing the third question.
Finally, the PRlacz mouse joins an expanding subfamily of mouse models that were designed to query the involvement of P in female reproduction in general and in mammary gland development in particular. Apart from accurately disclosing PR promoter activity in situ and representing a mouse platform with which to explore new E-and P-signaling pathways in the future, the PRlacz mouse validates our knockin strategy as a general genetic approach with which to position other genes, such as CRE recombinase (45) and the reverse tetracycline transactivator (46, 47), under the precise control of the PR promoter, such future PR knockins will be indispensable to unraveling the molecular mechanisms that underpin PR function in vivo.
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MATERIALS AND METHODS
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Targeted Insertion of the LacZ Reporter into the Murine PR Gene
A 7-kb genomic DNA fragment (129Sv derived) containing the 5' upstream-untranslated region and sequence extending from exon 1 to intron 3 of the murine PR gene (5) was used to construct the PRlacZ targeting vector. A bacterial-derived lacZ reporter gene (pD46.21) (48), harboring a nuclear localization signal and a novel HindIII site, was inserted between unique HincII-XhoI sites located within exon 1 of the mouse PR gene; the HincII and XhoI sites are positioned 356 bp and 724 bp downstream from the initiator codon ATGB for the PR-B isoform, respectively. The pD46.21 cassette was originally provided by Dr. Eric Olson (University of Texas Southwestern Medical Center, Dallas, TX). The above lacZ insertion step resulted in the attendant removal of a 122-aa fragment [aa 119 (leucine)-241(lysine)], which includes the initiating methionine (166) for the PR-A isoform (16). To enable drug selection, the neomycin resistance (neor) cassette, pgkneobpA, was cloned immediately 3' to the lacZ reporter; the pgkneobpA cassette was previously described (5). With a concomitant 366-bp deletion of PR sequence, the combined insertion of the lacZ and neor cassettes into exon 1 of the PR gene segregated the above 7 kb mPR genomic fragment into 5' and 3' arms of homology that were 1.2 and 5.5 kb in size, respectively. The herpes simplex virus thymidine kinase (MC-1 HSV-TK) gene (17) was cloned 5' to the 1.2-kb arm of homology with a transcriptional orientation opposite to both the neor and mPR genes; the MC-1 HSV-TK was a gift from Dr. Mario R. Capecchi, Howard Hughes Medical Institute Research Laboratories, University of Utah (Salt Lake City, UT). The cloning plasmid used in this vector construction was pSP72 (Promega Biotech, Madison, WI). Before electroporation into mouse ES cells, the PRlacZ targeting vector was linearized at the 3' end of the long arm of homology with the restriction enzyme Asp 718.
All ES cell culturing and manipulations before and after the electroporation step were preformed as previously reported (5). At a cellular concentration of 107 cells/ml, ES cells (AB2.2) were electroporated with 25 µg of the PRlacZ targeting vector in a volume of 0.9 ml at 230V and 500 µF; AB2.2 cells were kindly provided by Dr. Allan Bradley (Baylor College of Medicine, Houston, TX). Using 5' and 3' DNA probes located just outside the region of homology contained within the targeting vector, correctly targeted ES cells [1-(-2-deoxy-2-flouro-1-ß-D-arabino-furanosyl)-5-iodouracil and G418 resistant] were identified by Southern analysis. Male chimeras obtained from two-targeted ES cell clones were crossed with C57BL/6 mice with resultant germ-line transmission of the lacZ insertion; for the investigations described herein, mice with a mixed 129SvXC57BL/6 background were used.
Staining for ß-Gal Activity, Double Immunofluorescence, and General Immunohistochemistry
For ß-gal detection within mammary gland tissue, the inguinal gland (abdominal no. 4) was spread on a glass slide before fixation in chilled 2% paraformaldehyde (PFA), pH 7.4, for 2 h at 4 C; fixed tissue was subsequently thoroughly rinsed in three changes of PBS over a 90-min period. Tissues were then immersed in lacZ staining solution [1.3 mM MgCl2, 15 mM NaCl, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6.3H2O, 0.02% Nonidet P-40, 44 mM HEPES (pH 7.9), and 0.05% (wt/vol) 4-chloro-5-bromo-3-indolyl-ß-D-galactopyranoside] at 37 C in the dark, for at least 2 h and up to 16 h depending on the degree of color reaction. After three consecutive 30-min PBS washes, stained mammary tissues were processed for whole mount and/or sectioning; the mammary gland whole mount procedure has previously been documented (5). For sectioning lacZ stained mammary glands, tissues were serially dehydrated in 70100% ethanol, postfixed overnight in either Bouins fixative or 4% PFA, then washed in 70% ethanol, embedded in paraffin and sectioned at 5 µm; both whole mounts and sections were lightly counterstained with 0.1% Nuclear Fast Red. Intact embryos were used for whole-mount x-gal staining of mammary glands; for sectioning after staining, embryonic skin overlaying the upper ventral thorax was removed and briefly postfixed in 4% PFA, consecutively washed with PBS and dehydrated with ethanol, before being paraffin-embedded, sectioned at 5 µm, and counterstained with Nuclear Fast Red. Sex determination of embryos by PCR amplification of the testis-determining gene, Sry, was performed according to Gubbay et al. (49).
Although whole pituitaries (and sections thereof) were stained after the procedures above, ovarian tissue (without the fat pad) was embedded in OCT medium (Tissue-Tek, Elkhart, IN), frozen on dry ice, and cryosectioned at 20 µm. Ovarian cryosections were fixed for 15 min in 2% formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P-40 in PBS, then washed in PBS, and incubated overnight at 37 C in the x-gal staining solution described above; sections were subsequently washed, fixed and counterstained with Nuclear Fast Red.
To confirm colocalization of PR and ß-gal expression, PRlacZ mammary sections were incubated with a rabbit antihuman PR polyclonal antibody (1:100 dilution; DAKO Corp., Carpinteria, CA) overnight at room temperature. After PBS washes, sections were incubated with biotinylated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) for 1 h at room temperature before incubating with TRITC-conjugated streptavidin (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After a blocking step with goat antirabbit Fab (1:100 dilution; Jackson ImmunoResearch Laboratories, Inc.), sections were incubated with a rabbit anti-ß-gal polyclonal antibody (1:200; Cortex Biochemicals, San Leandro, CA) overnight at room temperature. Washed sections were incubated with biotinylated goat antirabbit IgG (Vector Laboratories, Inc.) before overlaying with streptavidin horseradish peroxidase [Tyramide Signal Amplification (TSA) Fluorescence Kit, NEL701 Perkin-Elmer Life Sciences (Boston, MA)] for 1 h at room temperature. Horseradish peroxidase was directly visualized by incubating sections with TSA-FITC substrate (1:50 dilution; TSA Fluorescence Kit) for 10 min at room temperature; slides were washed and mounted before microscopic examination. Using a Carl Zeiss (Jena, Germany) Axioplan 2 microscope equipped with epifluorescence and appropriate TRITC and FITC filters, digital images were captured using Metavue Software 4.6r9 (Universal Imaging Inc., Downington, PA); final image processing and assembly were performed using Adobe Photoshop version 6.0 (Adobe Systems, Inc., San Jose, CA).
Immunohistochemical detection of PR and ER expression and 5-bromo-2-deoxyuridine incorporation has been described previously (7, 39, 50); for ER immunofluorescence, a rabbit polyclonal antibody raised against aa residues 145159 of the human ER protein sequence (Geneka, Biotechnology Inc., Montréal, Canada) was employed using approaches reported by Seagroves et al. (39).
Images were captured using a color chilled 3CCD video camera (C5810, Hamamatsu Corp., Bridgewater, NJ) attached to a Carl Zeiss Axioskop microscope; photomontages were assembled using Adobe Photoshop.
Mice and Hormone Treatments
Mice were maintained in a temperature controlled (22 ± 2 C) room, 12-h light, 12-h dark photocycle and fed rodent chow meal (Purina Mills, Inc., St. Louis, MO) and fresh water, ad libitum.
Pituitary, Uterine, and Ovarian Studies
Pituitary PRlacz promoter activity was induced or suppressed by an overnight exposure to E (1 µg) or P (1 mg), respectively. For uterine hormonal stimulation, ovariectomized mice were administered daily E (1 µg) and/or P (1 mg) for 4 d described previously (26); the superovulation hormonal regimen was employed as reported (5).
Mammary Gland Studies
Mammary glands were dissected from mice at various stages of mammary gland development: virgin (5, 8, 12, and 16 wk), pregnancy [d 3 (early) and 18 (late)], day of parturition, lactation (d 7), and involution (d 7); for timed pregnancies, the morning of observing the vaginal plug was designated as d 0.5 of pregnancy. Mammary glands from male mice were taken at 16 wk of age and at embryonic d 14. For experiments described in Figs. 6
, 7
, and 9
, E (1 µg) and/or P (1 mg) (in sesame oil) were administered by daily intrascapular sc injections for 10 d, according to procedures reported previously. In the case of the 7-d lactating and involuting mammary gland, PRlaz expression was induced by an overnight exposure to E (1 µg).
To remove endogenous ovarian steroid hormones, bilateral ovariectomies were performed according to procedures outlined previously and were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and were in accordance with the procedures detailed in the Guide for Care and Use of Laboratory Animals (NIH Publication 85-23).
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ACKNOWLEDGMENTS
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The technical assistance of Shuangli Li is gratefully acknowledged; we thank Tiia Ponnio for initial instruction in the lacZ staining procedure and Drs. Daniel Medina and Jeffrey M. Rosen for critical reading of the manuscript.
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FOOTNOTES
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This study was supported in part by NIH Grants CA-77530-01 and HD-42311-01 (to J.P.L. and F.J.D., respectively) and by Department of Defense Breast Cancer Research Program IDEA Award DAMD 17-01-1-0138 (to J.P.L.).
Abbreviations: aa, Amino acid; E, estrogen; ER, estrogen receptor; ES, embryonic stem; FITC, fluorescein isothiocyanate; ß-gal, ß-galactosidase; hCG, human chorionic gonadotropin; neor, neomycin resistance; P, progesterone; PFA, paraformaldehyde; pgkneobpA, neor cassette; PMSG, pregnant mare serum gonadotropin; PR, progesterone receptor; PR-A and PR-B, two PR isoforms; PRKO, PR knockout; TEB, terminal end bud; TRITC, tetramethlyrhodamine isothiocyanate; TSA, Tyramide Signal Amplification.
Received for publication May 9, 2002.
Accepted for publication July 25, 2002.
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