functional rescue of stat5a-nuil mammary tissue through the

Vol. 9, 795-803, September 1998 Cell Growth & Differentiation 795

varying environmental conditions.

Functional Rescue of Stat5a-nuIl Mammary Tissue throughthe Activation of Compensating Signals Including Stat5b

Xiuwen Liu, Matte I. Gabbego, Gilbert H. Smith,Gertraud W. Robinson, and Lothar Hennighausen1

Laboratory of Genetics and Physiology, National Institutes of Diabetes,Digestive and Kidney Diseases, NIH [X. L, M. I. G., G. W. A., L H.], andLaboratory of Tumor Biology and Immunology, National CancerInstitute [G. H. S.], Bethesda, Maryland 20892

AbstractProlactin induces mammopoiesis and lactogenesisthrough the Janus kinase-signal transducers andactivators of transcription pathway, with Stat5a being aprincipal and obligate cytoplasmic and nuclearsignaling molecule. Mice from which the Stat5a genehas been deleted fail to develop functional mammarytissue during their first pregnancy. Lobuboalveolaroutgrowth is curtailed, and epithelial cells fail toprogress to functional differentiation. Here, weinvestigate whether the effect of Stat5a deficiency isrestricted to the epithelium and whether the gland hasthe capacity to activate alternative signaling pathwaysthat could restore development and function.Mammary gland transplant experiments showed thatStat5a-deficient epithelium does not differentiate inwild-type stroma, thus demonstrating a cell-autonomous role for Stat5a. The capacity of Stat5a-deficient mammary tissue to develop and secrete milkwas measured after consecutive pregnancies and withpostpartum suckling. Neither of these regimens couldindependently restore lactation. However, thecombination of several pregnancies and sucklingstimuli resulted in a partial establishment of lactationand an increase of Stat5b activity. These experimentsdemonstrate that the mammary gland has inherentplasticity that allows it to use different signals toachieve its ultimate purpose, the production of milk tonurture newborn offspring.

IntroductionLobubo-alveolar proliferation and differentiation in the mam-mary gland during pregnancy and lactation requires the co-ordinated action of steroid and peptide hormones (1). Inparticular, the Pr12 signaling pathway is critical for functional

Received 5/8/98; revised 7/22/98; accepted 7/23/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cete this fact.1 To whom requests for reprints should be addressed, at Laboratory ofGenetics and Physiology, NIH, Building 8, Room i0b , Bethesda, MD20892-0822. Phone: (301) 496-2716; Fax: (301) 480-7312; E-mail:[email protected] The abbreviations used are: PrI, prolactin; PrIR, Pri receptor� JAK, Januskinase; STAT, signal transducers and activators of transcription; wt, wild-type.

mammary gland development and differentiation (2). Bindingof Pri to the PrIR induces receptor dimerization and activa-

tion of JAK2 (3, 4). JAK2, in turn, phosphorylates transcrip-

tion factors that belong to the STAT family (3, 4). Phospho-

rylation of specific tyrosine residues confers DNA bindingactivity to STAT proteins and results in the transcriptionalactivation of genes containing IFN-y activation sites (TTC-NNNGAA). The importance of the PrI signaling pathway formammary development has been illustrated in mice in whichdifferent components of the pathway have been inactivated

(5-7). Both PrI (5) and its receptor (6) are required for ductal

branching and alveolar development and lactation. AlthoughPrI signals through the mitogen-activated protein kinase andJAK-STAT pathways, the former does not seem to function in

mediating differentiation in the gland. In the absence ofStat5a, alveolar proliferation is curtailed and, due to a lack ofepitheliab cell differentiation, dams fail to lactate (7). In mam-mary tissue of these mice, Stat5b activity is diminished. Theprotein sequences of Stat5a and Stat5b are 96% conserved.Although the DNA binding domains are very similar, substan-tial differences cluster in the COOH-terminal transactivationdomain (8). On the basis of transcriptional activation of a

synthetic reporter gene in transfection assays in tissue cub-ture cells, Stat5a and Stat5b display no functional differ-ences, suggesting similar if not identical biochemical profilesfor these two molecules. However, the in vivo studies in micesuggest that the presence of Stat5b cannot compensate forthe absence of Stat5a during their first pregnancy.

Although it is clear that the presence of Stat5a is required

for functional mammary gland development, several ques-tions pertaining to the mechanistic role of Stat5a during theproliferative phase of alveolar development and during dif-

ferentiation need to be addressed. Here, we investigated twofunctional aspects of the Stat5a defect. First, we determinedwhether the Stat5a defect manifests itself in the epithelium orstromal compartment of the gland. Using reconstitution ex-periments of wt and mutant tissue components, we showthat development of Stat5a-null mammary epithelium is also

curtailed when it is transplanted into wt stroma. This pro-vides formal proof of the function of Stat5a in the mammaryepithelium. Second, we explored the possibility that the de-fect can be overcome by multiple pregnancies, as is the casein PrIR +/- (hemizygous) mice (6). Third, we asked whetherother signaling pathways can compensate for the absence ofStat5a. Functional development of the mammary gland isdependent on several steroid and peptide hormones andgrowth factors that signal through a number of cytoplasmicand nuclear pathways. It is, therefore, reasonable to predictthat the lack of one of these pathways can be compensatedfor given the introduction of appropriate stimuli. Such a plas-ticity, if existent, would endow the organism to adapt to

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Fig. 1 . Whole mount analysis of ductal and alveolar morphogenesis. Stat5a-deficient (A and C) and wt (B and D) epithelia from mature virgin dams weretransplanted into cleared fat pads of wt virgin hosts. Ductal outgrowth and branching was evaluated after 8 weeks (A and B), and alveolar proliferation wasevaluated shortly after partunition (C and 0). Extensive ductal outgrowth and branching was observed with the wt (B) but not with the mutant epithelium(A). Solid arrows (A and B), branched epithelium. Open arrows, ample terminal end buds that were maintained in the mutant epithelium but less so in thewt. After delivery, mutant epithelium does not completely fill the fat pad and alveoli are small (C), whereas wt epithelium appears dense, indicating fullymature alveoli (D). Solid arrows (C and D), alveolar lobes. Scale bar, 5 mm.

ResultsAssociation of Stat5a Defect with Mammary Epithelium.Functional development of the mammary gland during pu-

berty and pregnancy is dependent on reciprocal interactionsbetween the stroma and epithebium. In the complete absenceof Stat5a, no functional mammary development was ob-

served during the first pregnancy. To dissect the robes ofStat5a in the epithelium and the stroma, we performed mam-

mary epithebial transplants. Removal of the mammary epi-

thebium from 3-week-old mice leaves a fat pad free of en-

dogenous epithelium, which, in turn, can be implanted with a

small piece of mammary epithelium from a syngeneic animal.

After several weeks, an entire epitheliab tree will grow fromthe transplant and penetrate the host fat pad. To investigatewhether the presence of Stat5a in the stroma contributes toductab elongation and branching, we performed mammary

gland transplants of null epithebium into wt stroma. wt recip-ients received a small piece of mutant epithebium into one fat

pad, whereas a control transplant from a wt or hemizygousanimal was transplanted into the contrabateral fat pad. Trans-

plants were harvested after 8 weeks, and whole mounts wereprepared to evaluate the morphology and extent of epithebialoutgrowth. Incomplete ductab outgrowth and a scarcity of

secondary branching was observed in the Stat5a-deficient

transplants (Fig. 1A). Transplanted control epithelium in the

contrabaterab gland fibbed the fat pad, and extensive second-

ary branching was observed (Fig. 1B). This experiment dem-onstrated that complete ductal morphogenesis in virgin miceis dependent on the presence of Stat5a in the epitheliab

compartment.To analyze the development of the epithelial transplants

in response to pregnancy hormones, wt hosts were mated,

and their mammary glands were harvested within 12 h of

parturition for histological and biochemical analyses. Al-

though wt epithebium completely fibbed the fat pad of wt

hosts (Fig. 1D), Stat5a-deficient epithebium remained un-derdeveboped, in that the alveoli were smaller (Fig. 1C).Histological evaluation of these whole mounts illustrated

that Stat5a-deficient epithelium had not fully infiltrated thefat pad and the alveoli had failed to expand, differentiate,and secrete milk (Fig. 2, C and D). In contrast, transplantedwt epithelium had completely filled the stroma, and thealveoli appeared fully secretory, with ample fat droplets

within the cytoplasm and lumen (Fig. 2, A and B). Theseresults support the notion Stat5a exerts its effects in ep-itheliab cells and is required for complete alveolar devel-

opment and function.

The chimeric glands consisting of transplanted mutantepithebium in wt stroma offered us the opportunity to ask

whether stromal cells contain active Stat5a. We analyzed wtand mutant epithelial transplants in wt host stroma for thepresence of Stat5a and its state of phosphorylation (Fig. 2E).

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Cell Growth & Differentiation 797

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Fig. 2. Histological and biochemical analyses of alveolar morphogenesis. wt (A and B) and Stat5a-deficient (C and D) epithelia grown in a wt host wereharvested after partunition, sectioned, and stained with H&E. Alveolar proliferation occurs both in the presence (A) and absence (C) of Stat5a. Although wttissue has a highly secretory character (B, arrows point to secretory vesicles in alveoli), less secretion is found in Stat5a-deficient alveoli (D). Specifically,wt alveoli are greatly expanded (B), and mutant alveoli remain small and condensed (D). lu, alveolar lumen; fat, fat pad. E, analysis of Stat5a expressionand phosphorylation in the wt and mutant tissue shown above. Immunoprecipitation of cellular protein was performed with anti-Stat5a antibodies, andWestern blots were performed with either antiphosphotyrosine (top) or anti-Stat5a (bottom) antibodies. lu, lumen; en, tissue from endogenous glands; +1-and -I- . gland with transplanted epithelium. Scale bars, 300 �m (in A and C) and 1 00 pm (in B and D).

As expected, tyrosine-phosphorylated Stat5a was present inan endogenous wt gland at parturition (Fig. 2E). The amount

of Stat5a in a hemizygous epithelial transplants was reduced

but appeared normally phosphorylated. In contrast, inStat5a-deficient epithelial transplants, we found only smallamounts of Stat5a, which were derived from the wt stroma.This Stat5a was not phosphorybated on tyrosine. Thus,Stat5a in the stroma is not activated by bactogenic hormonesduring pregnancy. This finding corroborates the results ob-

tamed from epithelial transplant experiments, namely that the

developmental defect in Stat5a-null mice is cell autonomous

and confined to the epithebiab compartment.Stat5a-mediated Alveolar Development during Preg-

nancy. The analysis of mammary development in Stat5a-deficient and wt mice during pregnancy identified a time

window during which Stat5a controls alveolar proliferation(Fig. 3). At day 8.5 of pregnancy, mammary tissues of Stat5a-null (Fig. 3A) and wt (Fig. 3E) mice had a similar appearance.The fat pad was laced with ducts, and occasionally, emerg-

ing immature alveoli were observed. Clear differences in


Fig. 3. Mammary development during pregnancy. Mammary tissue from wt (E-H) and Stat5a-null (A-D) mice was harvested at days 8.5 (A and E), 10.5(B and F), 1 2.5 (C and G), and 1 7.5 (0 and H) of pregnancy, sectioned, and stained with H&E.

alveolar development were observed at day 1 0.5 of preg-

nancy (Fig. 3, B and F). In contrast to the mutant mice, the

alveolar density in wt mice had increased. The discrepancy in

alveolar density widened throughout the progression of

pregnancy (Fig. 3, C and G). Just prior to parturition, the fat

pad of wt mice was mostly fibbed with secretory alveoli (Fig.3H) but was still sparse in the mutant mice (Fig. 3D). In

addition, although fat droplets as a clear sign of functional

differentiation emerged at this point in wt mice, no such

alveolar differentiation was observed in the null mice. These

experiments demonstrate that the shortage of alveoli in theStat5a-nulI mice was the cumulative result of insufficient

proliferation after day 8.5 of pregnancy.

Electron Microscopic Analysis of Stat5a-null Alveoli.Histological analyses had revealed that mammary alveoli

could develop to some extent in Stat5a-nulb mice but failed

to differentiate and form a secretory lumen. Electron mi-

croscopy was used to evaluate the ultrastructurab com-

partments (Fig. 4). A small but defined lumen was visible atday 1 8 of pregnancy, a network of Golgi apparatus was

present, and casein micebbes and lipid droplets were visible

in both the cytoplasm and lumen (Fig. 4). Although secre-

tory components and vesicles were present in the cyto-

plasm of Stat5a-nulb mammary epitheliab cells, their

amount was greatly reduced as compared to mammary

cells from control mice (data not shown). This is in agree-ment with our finding that milk secretion in Stat5a-nubI

mice is sharply reduced.

Effect of Suckling on Mammary Gland Developmentand Stat5b Activation. We had observed that the bevels of

activated Stat5b were greatly reduced in Stat5a-nuII mice at

parturition (7). Therefore, it is unlikely that Stat5b compen-

sates at this point for the absence of Stat5a. However, it has

been documented that local stimulation of mammary tissue

by active suckling is necessary for complete postpartum

alveolar development (9, 10). We, therefore, investigatedwhether continued postpartum suckling can further stimulate

functional mammary development in Stat5a-nuIl mice. Con-

tinued suckling of Stat5a-nulb postpartum females was

achieved by swapping litters between wt and mutant mice

every 12 h. Despite continued suckling, no significant struc-

turab or functional improvement of mammary tissue was ob-

served. Alveolar density remained bow (Fig. 5), and Stat5a-null dams were not able to feed pups even after 1 0 days of

continued suckling.

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Fig. 4. Electron microscopic analysis of Stat5a-defi-cient alveoli. Mammary tissue from Stat5a-null mice atthe time of parturition was subjected to electron mi-croscopy. Go, Golgi apparatus; nu, nucleus; Ip, lipiddroplet; mfg, milk fat globule. Arrows, casein micelles.

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The consequence of continued suckling on the status of

Stat5b phosphorybation was investigated. Phosphorylation of

Stat5b in mammary tissue of mutant mice was equally low at

days 1 and 3 after parturition (Fig. 6). In the three miceanalyzed at 9 days after parturition, Stat5b phosphorybation

was low in one mouse and significantly higher in two of them(Fig. 6). In those mice with increased Stat5b phosphorybation,

Stat5b protein bevels were also elevated. Although this ex-

periment did not provide formal proof that Stat5b phospho-

rybation is dependent upon continued suckling, it indicated

that continued local stimulation through suckling, in some

cases, resulted in an increased bevel of Stat5b phosphory-bation.

Multiple Pregnancies Combined with Suckling Can In-duce Functional Mammary Gland Development. In an at-

tempt to induce functional mammary gland development in

Stat5a-nubb dams, we introduced a regimen combined of

multiple pregnancies and continued suckling. After each

pregnancy, litters were swapped between Stat5a-null and wt

mice and allowed to suckle for 7 days. Subsequently, these

dams were bred, and the procedure was repeated. No signs

of milk were found in pups nursed by Stat5a-null dams after

the first and second pregnancy. However, after 4 days of

suckling after the third pregnancy, milk appeared in the

stomachs of the pups, and the dams were able to support

their litters. This suggests that the combination of continued

stimulation by systemic bactogenic hormones and local fac-

tors can functionally rescue the Stat5a-null mammary gland.

Histological analysis revealed a partial structural rescue of

Stat5a-deficient alveoli (Fig. 7). Alveoli of mutant mice were

small and condensed just prior to parturition during the third

pregnancy (Fig. 7A) but exhibited a partially expanded and

secretory appearance 3 days after parturition and extensive

suckling (Fig. 7C). In control mice, secretory vesicles and fat

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Fig. 5. Mammary development afterthe first pregnancy and 10 days ofsuckling. Mammary tissue from wt (Band D) and Stat5a-null (A and C) wasanalyzed at days 1 (A and B) and 10 (Cand D) after the first pregnancy.

Fig. 6. Activity of Stat5b inStat5a-null mice after extensivesuckling. Mammary tissue wasisolated from wt and Stat5a-nullmice after their first pregnancy atdays 1 , 3, 9, and 1 0 postpartum.Litters were swapped betweenwt and Stat5a-null mice every12 h to ensure continued suck-ling. Total mammary proteinswere immunoprecipitated (IP)with either anti-Stat5a (left) orStat5b (right) antibodies, sepa-rated in SDS-polyacrylamide

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protein in mutant mice at day 19 of the third pregnancy were

lower than in wt mice but similar at postpartum devebopmen-

tab stages (Fig. BA). Using Northern blot analysis, we couldfurther demonstrate that the amount of Stat5b RNA in

Stat5a-nubb mice surpassed that found in wt mice (Fig. 8B).

Stati and Stat3 bevels were slightly higher in Stat5a-nubb

glands than in those from wt mice (Fig. 8A).One marker for nonfunctional mammary tissue in Stat5a-

null mice is the reduced expression of the WAP gene (7). Thestructural and functional rescue of mammary development

after three pregnancies, followed by suckling, was mirrored

by the activation of milk protein genes. Expression of theWAP gene was bow during the third pregnancy but ap-proached levels seen in wt dams after parturition and pro-longed suckling (Fig. 8C).

DiscussionFunctional development of the mammary gland during preg-

nancy requires the intricate interaction between female en-

droplets were clearly visible at day 19 of pregnancy (Fig. 7B),

and fully expanded alveoli fibbed the fat pad after 4 days ofsuckling (Fig. 70). Although Stat5a-nubl mice can support a

bitter after three pregnancies, the pups have a reduced

growth rate. This suggests that the mammary tissue does not

function at full capacity. This is supported by the histological

picture. Although wt alveolar cells during lactation are thin

and line a dilated lumen (Fig. 7D), mutant alveoli do not

expand, and the epitheliab cells are cuboidab (Fig. 7C).

To evaluate whether the partial functional rescue was pos-

sibby induced by elevated bevels of other Stats, notably

Stat5b, we analyzed Stat protein expression and their acti-

vation patterns (Fig. 8A). Phosphorylation of Stat5b in

Stat5a-nuII mice at day 19 of the third pregnancy was bower

than in wt mice but was equal or higher at days 1 and 4 after

parturition. Elevated Stat5b activity suggests that this highly

conserved protein may partially compensate for the absenceof Stat5a in the establishment of partial lactation. We evab-

uated whether the increased Stat5b activity paralleled in-

creased bevels of Stat5b protein and RNA. Levels of Stat5b


Fig. 7. Mammary development after the three pregnancies and 10 days of suckling. Mammary tissue from wt (B and D) and Stat5a-null (A and C) micewas analyzed at day 1 9 of their third pregnancy (A and B) and 4 days after parturition (C and D). Arrows, alveoli. Scale bar, 1 00 �m.

docrine hormones, the mammary gland epithebium, and the

stroma. Developmental stimuli include steroid (estrogen,

progesterone, and hydrocortisone) and peptide (PL, PrI, and

oxytocin) hormones (1 1); suckling by the young, which main-

tains mammary function through the release of PrI and oxy-

tocin; and local factors that prevent involution (9, 1 0). The

analysis of knockout mice has shed bight on the different

genetic and biochemical pathways that control mammary

development (1 1). A particular emphasis has been placed on

the PrI signaling pathway, which proved to be essential for

ductal and alveolar morphogenesis, differentiation, and ac-

tation (2). Mice with deletions of the Pr!, PrIR, Stat5a, and

Stat5b genes have been generated and investigated. PrI-deficient dams were infertile, and pregnancy-mediated alve-

obar development could not be studied (5). However, it was

shown that the presence of PrI is required for ductab branch-

ing during puberty (5). Similarly, mice without a PrIR did not

maintain pregnancies (6). Dams with only one functional PrIR

allele displayed severely compromised alveolar proliferation

and failed to lactate after their first pregnancy (6). This sug-

gests that a threshold bevel of the receptor is required forfunctional development. In the absence of Stat5a, females

reproduced normally but failed to lactate because mammary

development was incomplete, and the secretory cells failed

to undergo functional differentiation (7). In contrast to Stat5a,

Stat5b was dispensable for mammary function (1 2). We have

now demonstrated that a functional mammary gland that

supports pups can develop in the absence of Stat5a. The

back of Stat5a can be bypassed, and a partially functional

gland can be restored after multiple pregnancies and exten-

sive suckling stimuli. Such plasticity in organ development is

achieved by compensation of signaling pathways, which

probably integrates Stat5b.

Epithelium-associated Effect of Stat5a. Our transplant

experiments have illustrated that the presence of Stat5a is

needed in the epithelial cells. Within the epithelium, Stat5a

controls two distinct layers of development, ductal and al-

veobar morphogenesis. On the basis of our transplantation

experiments, we conclude Stat5a signaling mediates ductab

branching during puberty. A similar reduction of branching

has been observed in PrI-null mice (5), suggesting that PrIand not growth hormone is the main signaling component of

this aspect of ductal development.

The Stat5 Family in Mammary Gland Development: IsStat5b a Doppelg#{228}nger of Stat5a? Stat5a and Stat5b ex-

hibit an overall conservation of 92% that is even more pro-

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Fig. 8. Activity of Stat5b in Stat5a-null mice after threepregnancies and 4 days of suckling. A, mammary tissue wasisolated from wt and Stat5a-null mice at day 1 9 of their thirdpregnancy (P19) and at days 1 (Li) and 4 (L4) postpartum.Litters were swapped between wt and Stat5a-null mice every12 h. Total mammary proteins were immunoprecipitated (IP)with anti-Stat5b (top) antibodies, separated in SDS-poly-acrylamide gels, and reprobed with antiphosphotyrosine andStat5b antibodies. +, wt tissue; -, Stat5a-null tissue. Thepresence of Stati and Stat3 was evaluated by immunopre-cipitation of mammary proteins with anti-Statb and anti-Stat3antibodies, followed by Western blots using anti-Statb andanti-Stat3 antibodies, respectively. B, Northern blot analysisof Stat5a and Stat5b ANA at day 19 of the third pregnancy(Lanes a and b) and at day 1 after the third pregnancy (Lanesc and d). The nylon membrane was probed with a DNAfragment that recognizes both Stat5a and Stat5b RNA. Lanesa and c, Stat5a +1- mammary tissue; Lanes b and d, Stat5a-I- mammary tissue. The bottom band in Lanes b and d(Stat5a-null tissue) is part of the Sta5b signal. Stat5b RNAcomigrates with 28S RNA, which results in the “splithng up”of this band (8). C, Northern blot analysis of WAP and a-ca-sein mANA at day 19 of the third pregnancy (Lanes a and b)and at day 4 after the third pregnancy (Lanes c and d). Lanesa and c, Stat5a +1- mammary tissue; Lanes b and d, Stat5a-I- mammary tissue.

nounced in defined functional domains (8). Such a similarity

could reflect overlapping and redundant functions in a spec-

trum of physiological responses elicited by activating hor-

mones, specifically, Prb. Extensive tissue culture experimentshave established that biochemical parameters of the two

isoforms and the physiological responses induced by them

are very similar. However, gene deletion experiments haverevealed distinct and separable functions for Stat5a (7) and

Stat5b (12).

During their first pregnancy, Stat5a-nuII mice do not de-

vebop a functional mammary gland (7), and both proliferation

and differentiation of alveolar tissue are compromised. Fur-

thermore, the bevel of Stat5b protein in Stat5a-nubb mammary

tissue is reduced, and little phosphorylation can be detected(7). These findings suggest that Stat5b may not be abbe tocompensate for the absence of Stat5a in the development of

a functional mammary gland. However, the combination of

three pregnancies and extensive suckling after each partu-

rition leads to partially functional mammary glands that could

support pups. Clearly, Stat5b is the prime candidate for the

signal that bypassed Stat5a and compensated for its loss.

Functional compensation of mammary tissue paralleled in-

creased bevels of Stat5b RNA and protein. Specifically, a

sharp increase in the tyrosine phosphorylated form of Stat5b

was detected after three pregnancies. This suggests that apersistent bactogenic stimulus can bead to further transcrip-tionab activation of the Stat5b gene and extensive phospho-rylation of Stat5b, which is mandatory for its activation.

Functional rescue of mammary function was associated with

a slight elevation of Stati and Stat3 protein bevels. However,at this point, it is not clear whether they also contribute in the

compensatory program.Deletion of the Stat5b gene demonstrated that this Stat5

isoform is not required for normal mammary development

and function (12, 13). These findings suggested that onlyStat5a is mandatory for mammopoiesis and lactogenesis. Onthe basis of Northern blot analysis of mammary tissue,

Stat5a is in excess over Stat5b. Therefore, at this point, it is

not clear whether the phenotypic differences seen in the

functions or quantitative differences. Although Stat5b is not

required for mammary development, it clearly is able andsufficient to substitute for Stat5a. While we were reviewingthis research, a report was published in which both theStat5a and Stat5b genes had been deleted in mice (13). Inagreement with our previous results (7), Stat5a-null mice didnot develop functional mammary tissue during pregnancy.

Mice from which both isoforms had been deleted failed tomaintain pregnancies and alveolar proliferation and differen-

tiation could not be studied. However, ductab elongation andbranching appeared to be normal in the combined absenceof Stat5a and Stat5b (1 3), suggesting a negligible role of Pri

in the puberal developmental phase.Lactation in hemizygous PrIR-null mice is also impaired

after their first pregnancy and can be restored after succes-sive pregnancies (6). We suggest that, in both the PrIR-nulland Stat5a-nubl mice, a persistent stimulation of the Pri path-way eventually results in the activation of its downstreamtargets and the development of a functional mammary gland.At this point, we cannot rube out that additional and yetundefined signaling pathways may get recruited and contrib-ute in the bobulo-alveolar proliferation and differentiation. The


activation of compensatory bypasses is probably not uniqueto the PrI pathways and has also been encountered in es-

trogen signaling (14). Deletion of the gene encoding thesteroid receptor coactivator-1 results in only partial hormoneresistance, probably because of the compensatory up-reg-ulation of TIF2, a member of the steroid receptor coactiva-tor-1 family (14). Compensation for the absence of a geneproduct provides enormous plasticity and flexibility to theorganism but, in turn, also provides a challenge to the re-searcher who may search for the function of a gene productin vain.

Materials and MethodsAnimals. For the suckling studies, wt and Stat5a-null dams were mated,and litters were swapped every 12 h after parturition. Specifically, age-matched litters from a wt and a Stat5a-null dam were switched at 8 a.m.and 8 p.m. for up to 10 days. This resuited in the continued suckling ofStat5a-null mice and maintenance of the suckling stimulus. Mammarytissue was harvested at necropsy. For each experiment, at least fourStat5a-null and four matched controls were analyzed.

Histology. Mammary tissue was harvested at necropsy. For wholemount examination, the tissue was fixed in Camoy’s solution for 2 h and

stained with carmine alum ovemight as described previously (15). Wholemounts were embedded and sectioned using standard methods. Mam-mary sections were stained with H&E and used for histological analysis.

Protein and RNA Analyses. Protein extraction from mammary tissue,immunoprecipitations, and Westem blothng were performed as describedpreviously (16). Antibodies for Stat5a, Stat5b, and phosphotyrosine havebeen described (16). Total cellular RNA was isolated and Northem blotanalysis was performed as described previously (17).

AcknowledgmentsWe thank Tony Wynshaw-Boris, Lisa Garrett, and Theresa Hernandezfrom the Human Genome Research Institute for providing animal spaceand initially breeding the mice.

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functional rescue of stat5a-nuil mammary tissue through the

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