Meyer Et Al. 2004

Steroids 69 (2004) 145159

Review

Sex steroids and growth factors in the regulation of mammarygland proliferation, differentiation, and involution

I. Lamote, E. Meyer, A.M. Massart-Len, C. BurvenichDepartment of Physiology, Biochemistry, and Biometrics, Faculty of Veterinary Medicine, Ghent University,

Salisburylaan 133, B-9820 Merelbeke, Belgium

Received 16 July 2003; received in revised form 10 December 2003; accepted 16 December 2003

Abstract

The mammary gland is subjected to major morphological and biochemical changes during the lactation cycle. It is therefore not surprisingthat this dynamic process is strictly controlled. The importance of the sex steroid hormones 17-estradiol and progesterone for normaldevelopment of the mammary gland was recognized several decades ago and has been unequivocally confirmed since. Furthermore, it isnow also established that the influence of sex steroids is not restricted to mammogenesis, but that these hormones also control involution.Another important regulatory role is played by growth factors that have been shown to modulate survival (epidermal growth factor,amphiregulin, transforming growth factor , insulin like growth factor, and tumor necrosis factor ) or apoptosis (tumor necrosis factor, transforming growth factor ) of mammary cells. However, the molecular mechanism underlying the influence of sex steroid hormonesand/or growth factors on the development and function of the mammary gland remains largely unknown to date. Also scarce is informationon the interaction between both groups of modulators. Nevertheless, based on the current indications compiled in this review, an importantfunctional role for sex steroid hormones in the lactation cycle in co-operation with growth factors can be suggested. 2004 Elsevier Inc. All rights reserved.

Keywords: Lactation cycle; Growth factors; Steroids; Mammary gland

1. Introduction

Over the past several decades efforts have been made todetermine the relationship between hormones and the mam-mary gland. Steroid hormones of the ovary and placentawere implicated very early as important stimulators of mam-mary gland development [1]. Since changes in developmentof the female mammary apparatus are particularly evidentduring gestation and lactation, these conditions were studiedmore intensively than others. All these efforts have led to animpressive number of original papers and excellent reviewson steroids and lactation biology. The aim of the currentreview was therefore not to provide the reader with com-pleteness with respect to the current knowledge of this topic.Rather, we wanted to contribute from an original point ofview putting the emphasis on what is now emerging as themissing link between systemic sex steroids and local factorsin the control of the mammary gland throughout the lacta-tion cycle. For this purpose, a comparison is made betweendifferent mammalian species to enlarge the scope from the

Corresponding author. Tel.: +32-9-2647321; fax: +32-9-2647499.E-mail address: [email protected] (E. Meyer).

traditionally favorite mammary gland animal model, the ro-dent (rat, mouse), to less intensively studied, but equallyinteresting species, such as ruminants (cow, goat, sheep).Some studies in human, pig, and dog are also included.For all species, the changes in the mammary gland duringgestation, lactation, and involution involve complex interac-tions between many hormones and cell types. Since most ofthese hormones induce the production of local factors, in-sight into the endocrine control of the mammary gland iscomplicated. The resulting interplay from systemic and lo-cal signals needs to be carefully considered to determine thebalance between proliferation, differentiation, and apoptosisof the different cell populations at all stages of the lactationcycle in the mammary gland.

2. Sex steroid hormones and the lactation cycle

The lactation cycle can be divided into different consec-utive stages, including, in chronological order, mammoge-nesis, lactogenesis, galactopoesis, and involution. Each ofthese phases is characterized by strict hormonal control. Thetraditional role of steroids and other hormones in different

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146 I. Lamote et al. / Steroids 69 (2004) 145159

mammalian species was already obtained from pioneer stud-ies in the 1950, but has been since refined using new tech-nologies like knock-out (KO) and transgenic animal models.In the following paragraphs, the current view on the hor-monal control of the mammary gland throughout the lacta-tion cycle is summarized starting from the classical conceptsto the most recent state-of-the-art reviews.

2.1. Mammogenesis

From birth to the onset of puberty mammary growth(mammogenesis) is minimal and proportional to that of thebody (isometric growth). This period is regarded as a qui-escent phase in the growth of the gland. A phase of moreactive mammary growth occurs around the time of pubertyand is characterized by a rapid extension and branching ofthe duct system (allometric growth). Thereafter, the degreeof mammary proliferation depends on the nature of the re-production cycle. The initial growth changes in the mam-mary gland in early gestation are related to the degree of de-velopment already attained during the reproduction cycles.In many species, further duct extension and duct branch-ing occur, followed by growth of lobules of alveoli. Duringgrowth the proportion of parenchyma to stroma increases,until eventually, the dense collection of lobes and lobules ofalveoli are separated only by septa of connective tissue. Itused to be thought that growth of the mammary parenchymabrought about by cell division was completed in the firsttwo-thirds of gestation and that the subsequent increase insize of the mammary glands was due to hypertrophy of theexisting alveolar cells and to the expansion of the alveoliwith secretion, but there is now clear evidence that cell di-vision occurs throughout gestation and continues into theearly stages of lactation [2].

The importance of sex steroid hormones for normal mam-mogenesis has unequivocally been confirmed. Throughoutgestation, proliferation of mammary epithelium is inducedby the sex steroid hormones 17-estradiol (E2) and proges-terone (P) [3]. E2 and P generally act as survival factors [4]

Fig. 1. Schematical representation of hormonal control of gestation, lactation and involution. During gestation, full lobulo-alveolar development takesplace under the continued stimulation of estrogen (E) and progesterone (P). In most of the positive mammary epithelial cells, ER and PR are colocalized,while in stromal cells only ER is localized in some species (only changes in ER expression are represented). ER and PR expression decrease throughoutgestation compared to the non-gestating animals where ER and PR expression are relatively high. Activation of ER probably induces proliferation ofmammary epithelium through stimulation of the expression of growth factors, which may be locally secreted by stromal cells (arrow). Activation of thePR-positive epithelial cells by P causes proliferation of the neighboring PR-negative cells (arrow). E can induce mammary PR expression via ER. Viceversa PR probably also interacts with ER (see detail). Although not fully characterized, epithelial progenitors (in blue) have been described in a largenumber of species. A dramatic decrease of P occurs around parturition and downregulation of PR expression is continued. ER is further downregulatedduring the transition of gestation to lactation, while during full lactation expression is again upregulated. A fall in E is not uniform in all species and wastherefore not indicated in this figure. In addition, rising levels of prolactin (PRL) and/or somatotropin (STH) are necessary for successful lactogenesis.PRL and STH induce functional differentiation through induction of transcription of milk protein genes. The importance of STH versus PRL is highlyspecies dependent (see detail). In early involution, ER expression decreases. Since an overlap between the periods of lactation and gestation exist insome species, general changes in E and P could not be indicated. The absence of PRL and STH is critical for mammary gland involution. Duringearly involution, apoptotic epithelial cells and a decrease in the expression of milk protein genes have been detected (see detail). In late involution PRexpression increases. Proteolytic degradation of the basement membrane by plasmin and matrix metalloproteinases (MMP) is initiated (see detail) andthe apoptotic process is continued. As progenitor cells are limited in their proliferation capacity, they need to be renewed probably after some lactationcycles. In the figure, extensive tissue degeneration is shown, although in a few species only isolated tissue degeneration is found.

in hormone sensitive tissues, such as the ovaries, uterus, andmammary gland [5]. Clinical studies on estrogen deficiencysyndromes in humans [6] have implicated estrogen in thenormal development of the breast. Definitive evidence forthe importance of estrogen signalling in normal mammarygland development has been obtained from experiments inmice. Firstly, castrated immature mice do not show ductalgrowth through the fat pad of the mammary gland, signify-ing that mammary ductal development is hormone depen-dent [7]. Secondly, the mammary glands of ovariectomizedmice are stimulated to grow by implanted estrogen pellets[8], and implants of pure anti-estrogens inhibit mammarygrowth in intact mice [9]. Finally, female estrogen receptorKO mice develop mammary glands with only vestigial ductspresent at the nipples [10]. Nevertheless, extensive prolifer-ation of the mammary gland in response to the ovarian sexsteroid hormones occurs only if the pituitary gland is in-tact. Ovarian hormones in the absence of pituitary hormoneshave little or no mammogenic activity. In both the rat andmouse, detailed studies have been carried out on the hor-monal requirements for mammary proliferation in the ab-sence of endogenous mammogenic hormones, i.e. after re-moval of the pituitary and ovaries, or pituitary, ovaries, andadrenals. These pioneer studies on the rat by Lyons and hiscolleagues and on the mouse by Nandi are well-known, andalthough some minor differences exist in the responses ofthe two rodent species, they both show that the hormonesrequired for duct growth are estrogen, somatotropin (STH),and adrenal corticoid. If P and prolactin (PRL) are added tothis combination, lobulo-alveolar growth is stimulated [2].More recent studies with PRLR knock-out mice, i.e. with-out functional allele, and PRL hemizygous mice, i.e. withone functional allele, showed that mammary developmentis essentially blocked at the stage of extended ductal out-growths during pregnancy but that a normal ductal networkis formed during puberty [11,12].

The molecular mechanism of the influence of sex steroidsand other hormones on mammogenesis is still far from com-pletely understood. It is generally accepted that sex steroids

I. Lamote et al. / Steroids 69 (2004) 145159 147

and growth hormones exert an influence on the mammarygland and that the genomic biological responses in the mam-mary gland are predominantly mediated by receptors, butit is surprising that most authors mention that specific re-ceptors for these hormones are only expressed at very lowand even undetectable levels in the mammary gland [13,14].However, recently Schams et al. [15] reported significant PR

and ER expression throughout gestation, lactation, and invo-lution in the bovine. Furthermore, no non-genomic effectsof steroids have yet been described in the mammary gland.For E2, the genomic biological responses in the mammarygland are predominantly mediated by the estrogen receptor (ER) and not by ER [3]. ER is localized both in theepithelial and stromal compartments of the mammary gland


[16]. In contrast, human [17] and heifer [18] stromal cellsapparently do not express ER. During ductal development,activation of ER induces proliferation of murine mammaryepithelium through stimulation of the expression of growthfactors (IGF-I), which are probably locally secreted by stro-mal cells (Fig. 1) (reviewed by Forsyth [14] and Hovey et al.[16]). Stromal factors mediating development during gesta-tion are postulated but not yet clearly demonstrated.

The biological responses of the mammary gland to P havebeen intensively studied by many research groups [19,20].Only genomic responses have been reported for P in themammary gland. In contrast with E2, these genomic effectsare mediated by two isoforms of the progesterone receptor(PR), PR-A and PR-B. The ratio in expression of both PRisoforms in the mammary gland is critical for the normal re-sponse to P and is therefore strictly controlled. Recent stud-ies (reviewed by Lydon et al. [20]) have demonstrated thatPR is selectively localized in the mammary epithelium andnot in the stroma. In addition, PR expression in the mammaryepithelium is strikingly heterogeneous; there are only fewPR-positive epithelial cells, which are apparently distributedat random throughout the majority of PR-negative epithelialcells (Fig. 1; gestation). This typical pattern of PR expres-sion appears to be evolutionarily conserved as it is com-parable in mouse and human. In both species, a paracrineepithelialepithelial signalling via PR is present (Fig. 1; ges-tation); activation of the PR-positive cells causes prolifera-tion of the neighboring PR-negative cells. Brisken et al. [21]speculate that Wnt proteins might function as the paracrinefactors that operate downstream of PR. Shyamala [19] andLydon et al. [20] independently suggest that a possible ex-planation for this heterogeneous PR expression is the asso-ciation of PR with a specific subtype of not fully differen-tiated, non-proliferating epithelial cells. These PR-positiveepithelial cells are presumed to remain in their progenitorstate during subsequent lactations. As for E2, it has beensuggested that activation of PR sensitizes the epithelial cellsfor proliferation following exposure to stromal growth fac-tors [19,20]. Mammary PR expression gradually decreasesat the end of gestation, and in the final phase of differenti-ation to secretory epithelium, PR expression is completelylost. Shyamala [19] suggests that this loss might even berequired to reach the stage of terminal differentiation.

Data obtained with ER-KO and PR-KO models confirmthat E mediated signalling via ER is essential for ductalmorphogenesis, while P signalling via PR is critical forlobulo-alveolar development. P is required for the transi-tion from ductal to lobulo-alveolar morphology. However,it should be noticed that under normal physiological con-ditions, E2 indirectly stimulates lobulo-alveolar architectureformation too because it can also induce mammary PR ex-pression via ER [22]. Vice versa, it has been shown in vitrothat PR can also influence the biological responses to ER,although further research is required to confirm this inter-action on the steroid receptor level. It should be remarkedthat ER and PR colocalize in 96% of PR-positive human lu-

minal mammary epithelial cells [23]. As in mice, cells thatexpress both ER and PR are nonproliferative [23,24], sug-gesting that E and/or P may stimulate adjacent ER/PR neg-ative cells to divide by a paracrine mechanism [23] (Fig. 1;gestation).

Shyamala et al. [25], Saji et al. [26] and Schams et al.[15] refined these observations respectively for PR, ER andthe combination of both receptors and demonstrated differ-ences in ER and PR mRNA and/or protein expression inthe mammary gland during gestation and lactation. Theseauthors found a relatively high mRNA expression of ERand PR in the mammary tissue of non-gestating heifers. Thishigh level was down-regulated at the onset of lactation andwas due to constantly high levels of P and increasing levelsof E2 during the second-half of gestation. The mRNA datawere confirmed by demonstration of the protein for ER andPR by immunohistochemistry with signals of staining of ep-ithelial cell nuclei. Additionally, an increased cytoplasmicPR staining of epithelial cells was obvious during lactogen-esis (Fig. 1).

2.2. Lactogenesis and galactopoesis

Once substantial lobulo-alveolar growth has occurred, thealveolar cells undergo organellar and biochemical differenti-ation and acquire the capacity to secrete milk. It is commonto differentiate between the initiation and the maintenanceof milk secretion. Upon parturition, withdrawal of P stimu-lates milk secretion. During the first few days after parturi-tion (production of colostrum and first milk) this process iscalled lactogenesis. Once milk secretion occurs, the suck-ling or milking stimulus promotes its maintenance, a processcalled galactopoesis [27].

During lactogenesis and galactopoesis, milk production iscontrolled by the lactogenic hormones PRL and STH. BothPRL and STH are essential for the transition from a prolifer-ative to a lactating mammary gland in all mammalian speciesstudied. Nevertheless, a quantitative distinction can be madebetween ruminants (cow, goat, and sheep) where the influ-ence of STH dominates over PRL during galactopoesis, andother species like rodents and humans where the influenceof PRL dominates over STH during galactopoesis as wellas during lactogenesis. In fact, STH is dispensable for lac-togenesis in mice and humans, as GHR knockout mice [28]and human dwarfs with mutations in either GH or GHR canlactate [29,30].

In general, basal levels of glucocorticosteroids are neces-sary to maintain metabolism and several specific hormoneactions and are also expected to play a permissive role dur-ing lactation. Nevertheless, there is no evidence that thesurge of glucocorticosteroids occuring around parturition isinvolved in lactogenesis. PRL and STH induce functionaldifferentiation through milk protein and fatty acid synthesis.The transcription of several milk protein genes like -caseinand whey acidic protein (WAP) significantly increases uponmid-lactation as compared to the onset of lactation. PRL


acts directly through mammary epithelial receptors and acti-vates various transcription factors. One of the key signallingmolecules activated by the PRL receptor is Signal transducerand activator of transcription 5 (Stat5). The basic role ofStat5 in the mammary gland is to mediate PRL signalling,while the PRL receptor in turn relies heavily on Stat5 tomediate its effects [31]. However, Miyoshi et al. [32] pro-vide evidence that Stat5 has also other functions than medi-ation of the PRL effect alone and vice versa that PRL sig-nalling is not strictly mediated by Stat5. In contrast to PRL,demonstration of functional receptors for STH in the mam-mary gland is scarce [33]. Nevertheless, it is certain that themammary gland is a site of STH production as well as STHaction [34]. STH is found in canine mammary secretions(particularly pre-partum and in colostrum) at concentrations1001000 times those in plasma, although the role of milkSTH remains uncertain [35]. Because milk STH concentra-tions are not correlated with fetal plasma STH and STH isnot absorbed intactly through the canine gastrointestinal tract[36,37]. The biological response to STH is thought to be in-directly mediated via the insulin-like growth factor I (IGF-I)system. However, there are many contradictory results sup-porting or refreshing this role for IGF-I which seems to bespecies dependent [35,38].

2.3. Involution

Upon fulfilment its functional purpose in the course ofnormal lactation, the mammary gland regresses gradually(gradual involution) and ultimately returns to a state of de-velopment only slightly in advance of that which existed atthe beginning of the first gestation. A much faster regressionof the mammary gland occurs following cessation of milk-ing of animals in early lactation (initiated involution) [39].The withdrawal of the suckling young (weaning) or the ces-sation of milking are both inducers of involution. One of thefirst steps in the dry period following weaning or cessationof milking is the interruption of the release of galactopoetichormones. As a result, milk stasis and a fast decrease in milksecretion and in the expression of genes responsible for milksynthesis, such as whey acidic protein, occur (Fig. 1). Nextto hormone withdrawal, another factor, feedback inhibitorof lactation (FIL), has been proposed to be involved in thereduction of milk synthesis and functional differentiation ofsecretory cells at milk stasis. It has been shown that FIL hasan inhibitory effect on protein synthesis. FIL has an imme-diate and direct effect on casein and lactose synthesis andlong term, probably indirect effects on cell differentiationby inhibiting synthesis of lactogenic hormone receptors onsecretory cells (reviewed by Knight et al. [40]).

Immediately after suckling was discontinued, apoptoticmammary epithelial cells have been detected in rodents.Nevertheless, during this initial phase, involution is still re-versible (Fig. 1, early involution). In the second phase of in-volution in rodents, proteolysis of the extracellular matrix isinitiated and the apoptotic process is continued (Fig. 1, late

involution (remodeling)) with an almost complete loss ofepithelial cells [41]. The response to weaning in ruminantsappears to be slower and less expressed than in rodents. Al-though somewhat intermingled, both phases of involutionare also present in ruminants.

The later involution phase is characterized by a generalincrease in expression of protease genes concomittant witha decrease of their inhibitors. The expression of plasmino-gen activators, which induce the formation of active plasminfrom plasminogen and are inhibited by STH, increases afterdrying off. This local increase of plasmin and plasminogenactivators is reflected in bovine milk as a result of the typi-cal gradual involution in cows [42]. Although plasmin(ogen)is important in the postlactational involution, Lund et al.[43] demonstrated in plasminogen knock-out mice that in-volution can proceed with some retardation in the absenceof plasminogen. Following activation, the plasmin enzymewill, in turn, activate members of the matrix metallopro-teinase (MMP) family. MMP include some collagenases andstromelysines and are essential catalysts in the proteolyticdegradation of the basement membrane and the extracellu-lar matrix of the mammary gland. Other marked increasesin expression are observed for gelatinase and tissue trans-glutaminase (Fig. 1, late involution) [44]. Although tissuetransglutaminase is not a protease, it also plays an importantrole in the apoptotic process. Its activation leads to the for-mation of a crosslinked protein scaffold in cells undergoingapoptosis. This protein scaffold may stabilize the integrity ofthe dying cells before their clearance by phagocytosis, thuspreventing the non-specific release of harmful intracellularcomponents and consequently inflammatory responses [45].It should be emphasized that although MMP are undoubtedlycentral to the process of mammary gland involution, apop-tosis in rodents begins prior to degradation of extracellularmatrix. Apoptosis may be triggered by other mechanismswhich effect downstream MMP expression. One possibilityis that changes in cellmatrix interactions occur. Prince et al.[46] suggest that the modulation of integrin ligand bindingactivity might play a role.

Mammary gland involution has been extensively studiedin rodents, and this model is often considered to reflect theprocess in other mammalian species as the specific knowl-edge on involution in other species is scarce. Nevertheless,quantitative inter-species differences are considerable. Espe-cially for ruminants, it seems not justified to assume that in-volution is similar to that in rodents because the sequence oflactation and gestation differs fundamentally between bothgroups of species. Indeed, in cows and goats, there is anoverlap between the periods of lactation and gestation, whilein rodents lactation is separated from gestation by a dry pe-riod. It should be remarked that although sheep are also ru-minants, they are non-gestating at drying off. The speed ofinvolution is slowest in ruminants and fastest in rodents, al-though both in rodents and ruminants, a limited number ofepithelial cells are programmed to die starting from peaklactation. The caprine mammary gland is exceptional as the


speed of involution is intermediate. Mammary tissue regres-sion in the bovine mammary gland remains limited even atthe end of the dry period prior to calving. In contrast to ro-dents where apoptosis and changes in gene expression areobserved to be major at 4 days after cessation of milking,minor changes occur only at 7 days after cessation of milk-ing in cows [47,48].

The quantitative apoptosis and gene expression data aresupported by morphological changes observed during invo-lution. While the alveolar structure has completely degener-ated after a dry period of 4 days in mice, it remains mostly in-tact at that time in the bovine mammary gland, and even aftera dry period of several weeks, only isolated tissue degenera-tion can be found. An important additional feature of rodentinvolution is the proteolytical degradation of the basementmembrane between stroma and epithelium (Fig. 1) whichstarts by an altered expression of MMP and their inhibitors.Analogous to apoptosis, basement membrane degradation isalready maximal at 4 days. In sharp contrast, the basementmembrane in ruminants is still largely intact at 7 days of dryperiod [47,48].

An explanation for this marked difference in cows is thata similar number of epithelial cells as in rodents will un-dergo apoptosis between two lactations, but that secretoryepithelial cells are not readily eliminated at the start of thedry period. This hypothesis is supported by data obtained inthe goat, the species characterized by an intermediate speedof involution combined with a similar degree of cell deathas rodents. In the caprine model, there is a partial survival ofthe secretory epithelium during the first period of mammaryinvolution.

A logical question is then which cells will survive andwhich cells will be replaced during the dry period? It is ev-ident from the subsequent reproductive and lactation cyclesthat the mammary gland possesses a strong regeneration ca-pacity. The presence of pluripotent stem cells and cell-linecommitted progenitors in the normal mammary gland hasbeen described by several authors in different species suchas the mouse [49,50], rat [51,52], human [53,54] and cow[55]. Nevertheless, the precise nature of these cells needs tobe further characterized using specific markers, especiallyin humans. Kordon and Smith [56] first suggested the exis-tence of a population of self-renewing and pluripotent stemcells in the mammary gland of mice. In addition, indicationsfor the presence of precursor cells with a limited differen-tiation potential that can only generate ductular or alveolarepithelial cells were also provided. As these precursor cellsare limited in their proliferation capacity, they need them-selves to be renewed by cells originating from the pluripo-tent stem cell population. It can therefore be postulated thatcandidate cells for renewal are the precursor cells that areresponsible for expanding and maintaining the number ofmammary epithelial cells in subsequent lactation. In thisway, involution prepares the mammary gland for an optimalmilk secretion capacity in the following lactation. Furtherresearch from Chepko and Smith [57] confirmed the initial

indications on the replacement of a subpopulation of olderepithelial cells by new epithelial cells with a higher secre-tory capacity, generated from precursor cells. The presenceof morphologically distinct stem cells and different types ofprecursor cells in the mammary gland was not only demon-strated in rodents (rat and mouse), but also in the human andthe cow. A similar hierarchy in progenitors was indepen-dently described by Stingl et al. [58] in human mammarytissue. Subpopulations of cells with stem cell characteristicswere also found in the bovine mammary gland by Hollandet al. [55] on the basis of differences in ultrastructural fea-tures and gap junction intercellular communication.

At the end of gestation, the new population of epithelialprogenitors likely differentiates under sex steroid hormonalinfluence as described above for juvenile secretory cells atthe onset of lactation (Fig. 1). In a recent publication, Wagneret al. [59] additionally demonstrated that this newly maturedepithelial cell population is not replaced during the followinglactation cycle in humans and retains a limited proliferationcapacity.

The causes and the detailed mechanism of the remodel-ing process are not exactly defined, but one of the primarystimuli identified in rodents and ruminants is the abruptwithdrawal of lactogenic hormones. In vivo, systemic lac-togenic hormone levels drop immediately after cessation ofmilking. The importance of the absence of PRL and STHfor mammary gland involution was first demonstrated in ro-dents [60] and has recently been confirmed in vitro for thecow [61]. The most pronounced effect on involution wasobserved in the absence of PRL. As PRL represses the ex-pression of the pro-apoptotic IGFBP-5 mRNA, PRL dele-tion leads to the halt of inhibition of IGFBP-5 expression inepithelial cells. In consequence, more IGF-I is sequesteredby IGFBP-5 and thus prevented from binding to its recep-tor and from suppressing apoptosis and delaying involution(Fig. 1) [61,62].

Data on the influence of sex steroids on mammary glandinvolution are scarce. Athie et al. [63] studied the effect ofexogenous E2 on the involution in cows, by examining thechanges in milk composition. Using this criterium, an accel-erated involution was found following administration of E2.However, the relevance of these observations for the physi-ological situation is not clear. In a second study, PR mRNAbecomes again detectable in glands from mice undergoinglactational involution after a period of being undetectableduring lactation [25]. However, it has also been suggestedthat the absence of PR expression is a potential key factorin the remodeling of the basement membrane between thestroma and the mammary epithelium during involution. In athird study by Schams et al. [15], the mRNA expression andprotein data for ER and PR show clear regulatory changessuggesting involvement of these receptors in bovine mam-mary gland involution. The increase of ER at 24 weeks ofinvolution and of ER 24 weeks after the end of lactationcan be interpreted as preparation of tissue for new mam-mogenic activity if specific signals are released. Although


reported in a limited number of studies, these data obtainedin different animal models clearly indicate that sex steroidhormones also modulate the mammary gland throughout theinvolution stage (Fig. 1).

3. Growth factors and the lactation cycle

An increasing list of local growth factors has beenshown to modulate survival and apoptosis in the mammarygland. Several of these proteins or polypeptides are alsocytokines. A stimulatory role in the proliferation and/ordifferentiation of mammary epithelial cells is suggested formost growth factors including epidermal growth factor, am-phiregulin, transforming growth factor , and insulin likegrowth factor. Tumor necrosis factor might play a dualrole, stimulating cell survival or death depending on thepresence or absence of other factors, whereas transforminggrowth factor has been found to be growth inhibiting andapoptosis inducing during several phases of the mammarycycle.

3.1. EGF

One of the main groups of growth factors affecting themammary gland is the family of epidermal growth fac-tors (EGF) including EGF, amphiregulin (AR), transforminggrowth factor (TGF), heparin binding EGF, betacellulin,and epiregulin. EGF family members exert direct mitogeniceffects and are therefore classified as typical survival factorsbased on observations with transgenic mice and from murinetumor cell line studies [6467]. EGFs all bind with varyingaffinities the epidermal growth factor receptor (EGFR), thefirst receptor of the ERBB-signalling network which com-prises four homologous receptor tyrosine kinases (ERBB14). In addition, heparin binding EGF, betacellulin, andepiregulin also bind another receptor of the ERBB-signallingnetwork namely ERBB-4 (reviewed by Pinkas-Kramarskiet al. [68]).

Studies in mice on ERBB expression and activatingprofiles revealed that signalling by EGFR (and possiblyERBB-2) is critical for ductal outgrowth. It is less clearwhether this receptor also functions in alveolar morphogen-esis and lactation. Since EGFR levels and phosphorylationwere shown to coordinately peak in late gestation andlactation, such a role is suggestive although it remains con-troversial (Table 1). This is not the case for signalling byERBB-2, -3, and -4, which is clearly important for alveolarmorphogenesis and lactation (reviewed by Troyer and Lee[69]).

Studies with AR- and/or EGF- and/or TGF-null miceconfirmed the fundamental role of EGFR in ductal mor-phogenesis and revealed a differential role for the EGFRligands. Mammary glands from adolescent AR null micedisplayed striking defects in ductal outgrowth. Additionalloss of EGF or TGF exacerbated the defect whereas mice

lacking only EGF and TGF had normal glandular arboriza-tion, underscoring the fundamental role of AR in ductalelongation [70]. AR is expressed exclusively by the ep-ithelium while EGFR would only be critical in the stroma.Taken together, activation of stromal EGFR by epithelial de-rived AR provides the epithelialstromal signal previouslypostulated to be necessary for ductal morphogenesis [69].

In order to understand the role of EGFR in ductal mor-phogenesis more in detail it will be necessary to identifyits critical downstream signalling partners. Obvious can-didate effectors are molecules previously implicated inductal morphogenesis, cell adhesion/migration or remod-eling of the extracellular matrix. These include integrinsubunits [70], the intracellular kinases, Src or FAK, andMMP. MMP are particularly attractive since several of themare expressed by stromal fibroblasts adjacent to advancingducts and are regulated by EGFR ligands in cultured cells[69].

Next to studies on receptor expression and activating pro-files, studies on the expression of EGFR ligands throughoutgestation, lactation, and involution in the mouse have alsobeen performed (Table 1) [6567]. The mRNA levels of allsubfamily members except for EGF decrease during ges-tation and disappear during lactation. In contrast, EGF ex-pression increases dramatically at the end of gestation andpeaks during lactation, with high levels found in human andmurine milk [71]. Inversely, EGF decreases during involu-tion when the expression of the other subfamily membersincluding TGF starts to increase again [72].

These observations also suggest a differential role for themammary EGF subfamily members in the lactation cycle. Itcan be postulated that TGF together with other EGF sub-family members might contribute more specifically to ep-ithelial proliferation during gestation as well as during thedry period, while only EGF would also play a role in thedifferentiation process during lactation. Data obtained fromstudies in rodent mammary cell cultures [73] are in accor-dance with this differential role for EGF family members.These data suggest that EGF is needed for proliferation andfor rendering cells responsive to lactogenic hormones, butthat following differentiation, EGFs role might be to preventapoptosis. It should be remarked that the survival growthfactor EGF has been associated with an increased expressionof the anti-apoptotic Bcl-2 family member Bcl-xL, whichcould be involved in the regulatory mechanism [74]. Despitethese observations, it is also possible that EGF expressionduring lactation relates specifically to its secretion in themilk rather than having a role in the differentiation processduring lactation.

That the role of the EGF subfamily is likely evolutionarilyconserved is supported by the fact that EGFR have beendemonstrated in the bovine and ovine mammary gland inaddition to that of the rodent (reviewed by Forsyth [14]).Moorby et al. [75] observed that a remarkable decrease inTGF binding capacity occured at the end of gestation andduring lactation in sheep.


Table 1Expression and/or blood concentration of growth factors in the mammary gland during the lactation cycle

Growth factor Stage of lactation cycle Reference

Gestation Lactation Involution

EGF family EGF(mRNA) Increase Peak values Decrease [71,72]Other EGF familymembers (mRNA)

Decrease Disappearance Increase [71,72]

EGFR Late gestation:peak values

Peak values [69]

IGF family IGF-I (mRNA) Decrease Low levels Increase [77]IGF-I (blood concentrations) After parturition: decrease;

during lactation: increase[78]

IGF-II (blood concentrations) Constant Constant Constant [79]IGF-IR Parturition: decrease [79]IGF-IIR Constant Constant Constant [79]IGFBP-1, -3, -6 (mRNA) Littleno expression Littleno expression [80,81]IGFBP-1, -3, -6 (bloodconcentrations)

High IGFBP-3 High IGFBP-3 [80,81]

IGFBP-2, -4, -5 (mRNA) Increase [82]TNF TNF (mRNA) Increase Decrease [93]

TNF (protein) Increase High level [93]TNFR subtype p55 (mRNA) Early lactation: peak values [90]TNFR subtype p75 (mRNA) Increase [90]

TGF TGF1 (mRNA) Decrease Very low levels High values [100,102,104]TGF3 (mRNA) Increase Very low levels [104]TGFR (mRNA) Very low levels High values [100,102]

3.2. IGF

The insulin-like growth factor (IGF) family of ligands(IGF-I and IGF-II), binding proteins (IGFBP 16), and re-ceptors (IGF-IR and IGF-IIR) play pivotal roles in growthand development of the organism. The precise role of IGFin the mammary gland is complex and not yet fully eluci-dated. There are indications that IGF-II would play a minorrole compared to IGF-I, as it is, for instance, not even ex-pressed in the human breast [76]. Nevertheless, the synthesisof IGF-I in the mammary gland has been described in manyspecies, and IGF-IR and IGF-IIR have been detected in themammary gland as well. Moreover, it has been shown thatIGF-I is a typical survival factor in the mammary gland. Forinstance, Amundadottir et al. [67] demonstrated that mam-mary tumor cells overexpressing pro-apoptotic proteins sur-vive in the presence of IGF-I. The activity of IGF-I is con-trolled by a family of specific binding proteins, the IGFBP.Some IGFBP members induce, while others inhibit the stim-ulatory effect of IGF-I and are thus associated with cell sur-vival or death, respectively.

The mammary expression and blood concentrations ofIGF, IGF-R, and IGFBP in the mammary gland have beenexamined during the lactation cycle in several species(Table 1). The highest expression levels of IGF-I are de-tected in nongestating heifers. There is a tendency forIGF-I levels to decrease during gestation, relatively lowlevels are found during lactogenesis and lactation, and anup-regulation occurs during involution [77]. These expres-

sion levels are not fully paralleled by the IGF-I concen-trations found in blood. Ronge et al. [78] showed that theIGF-I concentration in cows decreases significantly afterparturition, followed by a gradual increase as lactation per-sists. Examination of the IGF-IR during the lactation cycleshows that the number of IGF-IR declines at parturition, achange that coincides with decreases in the blood level ofits ligand. In contrast, IGF-II and IGF-IIR remain largelyunchanged in cows (reviewed by Baumrucker and Erondu[79]). During lactation and involution, there is little or noexpression of IGFBP-1, -3, or -6 mRNA in rat, while ithas been shown that IGFBP-3 blood concentrations arehigher during both the prepartum period and involutionin ruminants [80,81]. The latter pattern fits well with thehypothesis that IGFBP-3 has a stimulatory effect on IGF-Iduring involution. During postlactational involution, thereis also a four-fold increase in IGFBP-2 mRNA and six-and 10-fold increases in the expression of IGFBP-4 and -5mRNA and protein within 24 h after weaning in rodents.Increased expression of IGFBP-5 correlates with apoptoticcell death in other tissues as well (reviewed by Rosfjordand Dickson [82]). Baumrucker and Erondu [79] postulatethat there is an important species difference in IGFBP func-tion. IGFBP-5 appears to be important in rodent mammarygland involution, while IGFBP-3 exhibits the greatest con-centration changes during involution in the bovine species.However, LeRoith et al. [83] also demonstrated the im-portance of IGFBP-3 in rodents. These authors observedthat transgenic mice expressing either IGF-I or IGFBP-3 in


mammary tissues produced milk, but had smaller alveolithan non-transgenic mice. After weaning, the mice express-ing either IGF-I or IGFBP-3 failed to fully undergo tissueremodeling, had decreased postlactational apoptosis, andtheir mammary glands retained enlarged lumens [83,84]. Asbinding proteins are expected to inhibit IGF activity, theseobservations may appear contradictory. However, IGFBP-3has been shown to retain active IGF by prolonging itshalf-life [8386]. It can therefore be suggested that the in-hibition of postlactational involution observed in IGFBP-3transgenic mice was due to a local accumulation of IGF-Iin the mammary gland. This was also indicated by studieswith transgenic mice overexpressing IGF-I des, a natu-rally occurring variant of IGF-I that lacks three N-terminalamino acids with a much lower binding affinity for bindingproteins. Transgenic mice overexpressing IGF-I des havealtered ductular and lobular morphology, similar to miceoverexpressing IGF-I, suggesting again that IGF-I is theactive factor that inhibits postlactational involution and thatIGFBP-3 is prolonging the half-life of this survival factor.

IGF-I is also called somatomedin (A and C) to indicatethat it mediates the STH-action. Indeed, the initial focus ofanimal scientists on the IGF system was brought about by aneffort to explain the galactopoetic effect of bovine STH [87].Because STH receptors were not found on lactating bovinemammary epithelial cells [38], the IGF were naively thoughtto be the active endocrine compounds that directly stimu-late milk production [79]. An increase of plasma IGF-I and-II after STH administration in lactating cows has been ob-served. IGF-I, but not IGF-II levels, also increased in milk.The question remains whether the ST-induced increase ofIGF-I could stimulate milk secretion (i.e. endocrine behav-ior) in lactating ruminants. Prosser et al. [88] infused IGF-Iinto the pudic artery of lactating goats and demonstrated anincrease in mammary blood flow and milk secretion. Themost obvious effect occurred early in the homolateral gland(direct action). In the heterolateral gland, the effect wasless pronounced and delayed by a few hours. It was furtherdemonstrated that intravascularly administered IGF-I can betransported from blood to milk across the secretory epithe-lium, probably using receptor internalization. This is an in-dication that the increased levels of IGF-I in milk after STHadministration can originate from extramammary sources.

To understand the role of IGF in the proliferation anddifferentiation of the mammary gland more clearly, down-stream signalling molecules needs to be elucidated. In gen-eral, IGF-IR acts through two primary cascades, the mitogenactivated protein (MAP) kinase and phosphatidyl-3-kinase(PI3-K) kinase pathways. The ultimate targets of the MAPkinase and PI3-K kinase cascades include members of theEts and forkhead transcription factor families. Regulation oftranscription factors provides a mechanism by which IGFmediates a proliferative and differentiative effect. However,it should be remarked that this mechanism is not specific forthe mammary gland [89].

3.3. TNF

Another player in the mammary regulatory network is tu-mor necrosis factor (TNF). TNF is mostly known as aninflammatory cytokine, but it has pleiotropic effects. TNF re-ceptors (TNFR) have been demonstrated in most tissues in-cluding human and rat mammary cells [90,91]. Basolo et al.[92] demonstrated the presence of TNF mRNA and proteinin human mammary epithelial cells. TNFwas first reportedas a potential regulator in the context of mammary prolifera-tion and differentiation in 1992 [93]. In an in vitro rat mam-mary gland model, TNF was found to stimulate epithelialcell proliferation both in the presence or absence of EGF. Itshould be remarked that in the paper from Dollbaum et al.[91], no stimulatory effect of TNF was observed when us-ing colony number as a parameter for the evaluation of pro-liferation. However, Ip et al. [93] compared colony numberwith cell number and found that while there was no increasein colony number, there was a systematic increase in cellnumber. This suggests that TNF is stimulating prolifera-tion of a selected colony population. TNF also stimulatesdifferentiation in vitro but only in the absence or upon de-ficiency of EGF. Remarkably, using a basement membraneof inferior quality in the in vitro model, TNF induces theformation of exquisite multi-lobularductal organoids veryreminiscent of the in vivo rat mammary gland during lacta-tion.

These morphological observations were complementedwith data on the functional differentiation of the mammaryepithelium, which was evaluated by the casein production.TNF inhibits casein production in the presence of EGF, butstimulates it in the absence of EGF in a concentration depen-dent manner. Varela et al. [73] have shown that TNF doesnot require the EGFR for its action on rat mammary epithe-lium and that the TNF and EGF mitogenic actions in themammary gland are mediated by independent pathways, al-though co-operativity may occur under some circumstances.It is not yet clear whether the mammary effects of TNFare mediated through a direct or an indirect mechanism ofaction. Ip et al. [93] speculate that one indirect action ofTNF could be to induce the expression of TGF.

How all these in vitro observations can be related to thephysiological situation was further studied by Varela and Ip[90]. Changes in the TNF and TNFR mRNA and proteinlevels were followed throughout the lactation cycle in the rat(Table 1). The obtained results are in accordance with thedata from the initial study from Ip et al. [93] and stronglysuggest a potential role for TNF in mammary epitheliumproliferation and morphogenesis during the lactation cycleof rats. During gestation there is a pronounced increase ofTNF at mRNA and protein expression levels, which prob-ably inhibits casein production until the onset of lactation.Throughout lactation, TNF mRNA levels decline, but ahigh expression of the TNF protein is observed. A possibleexplanation for this difference could be that the protein has


a longer half-life and persists even when the correspondingmRNA has already disappeared. A marked differential ex-pression was also observed for the two TNFR subtypes p55and p75. As for the TNFR, the p55 subtype mRNA peaksin early lactation and act as the sole mediator of prolifera-tion. In contrast, mRNA of the p75 TNFR subtype increasessteadily throughout lactation and is responsible for func-tional stimulation as reflected by casein production. Thesefindings in rat are not always in accordance with humanstudies, which are moreover seemingly contradictory. Ba-solo et al. [92] observed TNF in human mammary epithe-lial cells, while Miles et al. [94] localized TNF and TNFRin the mammary stroma, and Pusztai et al. [95] failed to de-tect both proteins in normal human breast tissue althoughthe p55 TNFR subtype was occasionally expressed in thestroma.

Is TNF also involved in mammary gland involution? Anactive role for TNF in this mechanism seems contradic-tory with its proposed function as a growth and differenti-ation factor. However, Varela and Ip [90] hypothesize thatthe well-known apoptosis-inducing action of TNF as ob-served in many other cell types might be inhibited in themammary gland by a yet unidentified protein in the otherstages of the lactation cycle and is no longer present at theinvolution stage. A link between TNF and MMP produc-tion was furthermore suggested [73] and has recently beenconfirmed in detail. The latter authors show that the secre-tion of MMP-9 is stimulated by TNF in vitro and postulatethat it may play a role in extracellular matrix degradationand subsequent controlled invasion of the stroma that occursupon mammary remodeling.

It should be remarked that the trigger for the release ofTNF and its exact source in the mammary gland remainlargely unknown to date. One possibility is that interstitialcells, such as leukocytes, found in the mammary gland serveas a source for TNF. Indeed, lymphocytes have been re-ported to aggregate in the mammary gland during gestationand lactation [96], and the proportion of macrophages wasreported to be greatest in the periparturient period in thecow [97,98]. Interestingly, not only the number of mammarymacrophages, but also their TNF production capacity wassignificantly increased with respect to their monocyte coun-terparts in blood.

3.4. TGF

A last group of cytokines that have been demonstrated inthe mammary gland of different species, such as the mouse[99], the sow [100], ruminants, i.e. the cow [101], and thegoat [102] are the members of the transforming growth fac-tor (TGF1, 2, and 3) family. In contrast to the othergrowth factors, these cytokines have been classified as localapoptosis inducing or growth inhibiting factors (reviewedby Rosfjord and Dickson [82]). The detailed mechanism ofTGF action is not yet known. While TGF regulation ofgene expression patterns of cell cycle elements such as cy-

clins and cell adhesion elements such as integrins have beenreported in mice, further research on the exact function ofthe TGF targeted genes in growth inhibition will increaseknowledge of the mechanisms by which TGF mediates itscellular effect [103].

In the mammary gland, a considerable amount of evidencehas accumulated indicating that TGF plays a critical roleduring several phases of the mammary cycle. TGF regu-lates growth and patterning of the mammary ductal tree inthe virgin mouse. During gestation, TGF is required foralveolar development and functional differentiation, while atthe same time inhibiting secretion of milk proteins. At par-turition this inhibition is lifted, permitting initiation of lac-tation. During the dry period, TGF was found to supportremodeling of the mammary gland [104].

Patterns of expression of different TGF isoforms duringthe mammary cycle have been examined and provide inter-esting insights into the action of TGF in mice (Table 1).Substantial expression of TGF1 and TGF3 was found inall stages of mammary development with the exception oflactation. TGF3 was substantially increased during gesta-tion, falling to negligible levels immediately following par-turition. TGF1 expression was strong in the virgin animalduring ductal development, declined during gestation, dis-appeared during lactation, and increased during involution[104]. In two comparable studies on the mammary glandof goats and sows, respectively, the expression of TGF1and its receptor (TGFR type III or -glycan) was moni-tored throughout the lactation cycle [100,102] (Table 1). Themammary expression of TGF1 and TGFR increases sig-nificantly from early lactation (very low levels) to the dryperiod (peak values) in the lobulo-alveolar tissue of bothspecies. In accordance, TGF family members are not foundin porcine milk [105].

During ductal development, accumulations of TGFaround mammary tissues are probably associated with theavoidance behavior of growing end buds, whose turning de-termines the pattern of interductal spacing. TGF1 inducesthe synthesis of stromal/extracellular matrix and basementmembrane proteins, which trap TGF, creating a locallynonpermissive environment for growth, resulting in cessa-tion of DNA synthesis and regression of the end bud [104].High TGF3 expression has been reported in the murinemammary gland during gestation where it was stated toinhibit the translation and probably also the secretion ofcasein [106] as has been postulated above for TNF. Alve-olar cells expressing TGF3 are thus fully prepared forlactation, but are inhibited from synthesizing and secretingabundant milk proteins. At parturition the level of TGF3drops abruptly, presumably permitting full expression ofthe lactational phenotype [104].

Wareski and Motyl [100,102] postulate that the TGFproteins are involved in the induction of programmedcell death during mammary involution in the caprine andporcine species. Although the specific mechanism of thisprocess was not investigated, an increase after TGF


administration in the epithelial Bax (a pro-apoptotic Bcl-2family member)/Bcl-2 (anti-apoptotic) ratio and in caspase-3activity was found. The ratio of pro- versus anti-apoptoticBcl-2 family members is essential in regulating the balancebetween survival and death in the mammary gland [107].A previous study in mice mammary epithelial cells showedthat PRL inhibits TGF1 transcription, which may explainthe low TGF1 mRNA and protein synthesis during lacto-genesis and galactopoesis as well as the TGF1 increase inlate lactation and the dry period that was also measured inthe sow.

4. Interaction between sex steroid hormones andgrowth factors

Cross-talk between receptors and their signalling path-ways has been shown to play a critical role in variouscellular responses to ligands. Such cross-talk may occurbetween receptors within the same family, such as theEGFR and IGF-IR, which are both tyrosine kinase recep-tors, or between different families, such as nuclear steroidreceptors ER and PR and IGF-IR. This last interaction hasbeen frequently described in mammary cells, but as mostof these experiments were exerted with breast cancer cells,they need to be confirmed in the normal mammary gland(reviewed by Hamelers and Steenbergh [108]).

Estrogens and IGFs act as mitogens promoting cell pro-liferation in the mammary gland. Originally, it was con-sidered that these agents manifest their mitogenic actionsthrough separate pathways, but a growing body of evidencegathered over the last decade suggests that the IGF- andestrogen-mediated signalling pathways are intertwined.

E2 has been shown to enhance IGF signalling at multi-ple levels (long-term and rapid effects). The expression ofIGF-IR and IGFBPs was found to be up-regulated by E2[109,110]. Huff et al. [111] and Cohen et al. [112] reportIGF-I mRNA and protein expression in MCF-7 cells, whichare up-regulated by E2, TGF, and EGF. However, it re-mains unclear whether breast cancer cells secrete IGF-I, asother groups [113115] have reported that MCF-7 cells donot express IGF-I. Next to these long-term effects, rapid ef-fects of the liganded ER on the IGF-IR could also be ob-served in transformed cell lines, but not in the cancer celllines studied by Hamelers et al. [116]. In the presence ofER, but not ER, E2 rapidly induced phosphorylation ofthe IGF-IR [117]. Vice versa, several studies have demon-strated that IGF signalling as well as signalling of other sur-vival factors, like EGF, result in the transcriptional activa-tion of ER [118122]. Co-administration of E2 and growthfactors to cells has been shown to result in an additive effecton the expression of endogenous estrogen-regulated genes[122]. Finally, IGF-I and E2 have been shown to synergis-tically stimulate proliferation of various cancer cell lines[114]. In analogy with the influence on IGF, estrogen wasreported to synergize with EGF in stimulating cell prolifer-

ation by upregulating EGFR [123]. A role for EGF in medi-ating estrogen induction of end bud formation and expres-sion of PR has been proposed [124]. EGFR is also believedto be a prominent downstream effector of estrogen action inseveral tissues.

In comparison to E2, few studies describe the interactionbetween P and growth factors. Moreover, in analogy withE2, the studies on P-associated growth factors were carriedout in mammary tumor cells and await confirmation in thenormal mammary gland. P induces the expression of severalgrowth factors via its PR. Since studies suggest a role for PRas well as for EGFR in ductal morphogenesis, these variousobservations support the view that EGFR is an essential me-diator of hormone action in the adolescent mammary gland[69].

Complementary to these data, which were mostly ob-tained from experiments with cancer cell lines, are the recentresults from Schams et al. [15] providing strong evidencethat interactions between ER and PR and TGF and IGF-Ialso occur in normal mammary gland tissue. These authorsdemonstrate that the mRNA expression pattern of some pro-liferative growth factors, such as TGF and IGF-I, in thebovine mammary gland during development and functionis comparable for ER and PR expression. The presenceof high ER, ER, and PR levels in the bovine mammarygland before the onset of lobulo-alveolar development andsignificantly lower levels during gestation and lactogenesis,suggests an important functional role for the initiation oflobulo-alveolar development, possibly in co-operation withthe proliferative actions of growth factors [15].

It should be remarked that additional evidence on thecross-talk between sex steroid hormones and other local fac-tors than IGF and EGF in the mammary gland is not onlylimited but also highly suggestive.

Sordillo et al. [98] postulate that local TNF productionmight be under steroid hormonal regulation, and Ip et al.[93] have suggested that the inhibitory effect of TNF oncasein production might be in concert with P. The meansby which TGF3 levels are regulated are not known, butregulation by changes in reproductive hormones such as P,ST, and PRL are possibilities [104]. Finally, it is not yetclear whether there is a link between the mammary effects ofTGF and TNF during involution, but it has neverthelessbeen suggested that hormonal influences could modulate theexpression of both cytokines [90,125]. Further research onthis topic is therefore clearly warranted.

5. Conclusion

The well known importance of the sex steroid hormonesE2 and P for normal mammogenesis has unequivocally beenconfirmed and refined by data obtained with KO and trans-genic animal models. These latter studies have establishedthat ER mediated signalling is essential for ductal morpho-genesis, while PR signalling is critical for lobulo-alveolar


development. ER and PR expression has also been deter-mined in the mammary gland during the lactation cycle andthe regulatory role of these steroid receptors and their lig-ands can now be expanded from mammogenesis to all otherphases including lactogenesis, galactopoesis, and especiallyinvolution. These data also suggest that mammary prolif-eration and differentiation are perfectly counterbalanced bycell death throughout the lactation cycle.

In addition to the regulatory role of sex steroid hormones,an increasing list of local growth factors has also been shownto modulate survival and apoptosis in the mammary gland.A stimulating role in the proliferation and/or differentiationof mammary epithelial cells is suggested for most growthfactors including EGF, TGF, AR and IGF. The cytokineTNF might play a dual role, stimulating survival or celldeath depending on the presence or absence of other factors,whereas TGF has been found only to be growth inhibitingand apoptosis inducing. For most of these growth factors,the expression patterns of their receptors were also moni-tored during the lactation cycle. These data reveal cross-talkbetween the ER and PR and the receptors for some growthfactors, suggesting an important functional role for estrogensand P in the lactation cycle in co-operation with these growthfactors. Nevertheless, the cross-talk between sex steroid andgrowth factor receptors has not extensively been reported.Moreover, the interaction with the IGF-IR and EGFR wasdescribed in breast cancer cell lines and still needs to beconfirmed in the normal mammary gland. In conclusion, itremains a challenge for future research to further unravel theinteraction between sex steroid and growth factor signallingpathways in the mammary gland.

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Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involutionIntroductionSex steroid hormones and the lactation cycleMammogenesisLactogenesis and galactopoesisInvolution

Growth factors and the lactation cycleEGFIGFTNFalphaTGFbeta

Interaction between sex steroid hormones and growth factorsConclusionReferences

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Meyer Et Al. 2004