Local IGF-I Axis in Peripubertal Ruminant Mammary Development

Journal of Mammary Gland Biology and Neoplasia, Vol. 5, No. 1, 2000 Local IGF-I Axis in Peripubertal Ruminant Mammary Development R. Michael Akers, 1,5 Thomas B. McFadden, 2 Stig Purup, 3 Mogens Vestergaard, 3 Kristen Sejrsen, 3 and Anthony V. Capuco 4 The regulation of mammary growth and development in heifers is accomplished by complex interactions of hormones, growth factors, and extracellular matrix molecules. Many of these growth stimulators are believed to be locally produced in the mammary gland and to be affected by developmental and nutritional status. Although estrogen and growth hormone are considered critical to pubertal mammogenesis, results summarized in this review suggest that IGF-I 6 and IGF binding proteins are especially important locally-produced growth regulators in peripubertal ruminants. This assertion is supported by studies of ovariectomized heifers, in which increased stromal IGFBP-3 and reduced IGF-I correspond with a failure of udder development. Similarly, reduced mammary development with overfeeding coincides with reduced mitogenic activity of mammary tissue extracts and altered concentrations of IGF-I and IGFBPs. In vitro studies convincingly demonstrate that much of the mitogenic activity of mammary extracts or serum can be attributed to IGF-I and that alterations in IGFBP-3 modulate its effectiveness. Thus by analogy to second messenger mechanisms of action for protein hormones, local mammary-derived growth factors likely explain many of the effects attributed to the classic mammogenic hormones. KEY WORDS: Bovine; ruminant; IGF; mammary; paracrine; growth factors. INTRODUCTION Classical endocrine-ablation-hormone replacement studies with rodents in the 1940s and 1950s confirmed requirements for the secretions of the pituitary and ovary in mammogenesis (1,2). Growth hormone (GH) and estrogen especially were deemed critical for pubertal ductal development, and synchronous secretion of estrogen and progesterone after conception essential for final duct elongation and lobuloalveolar formation. Surgical complications have made similar studies difficult in economically important 1 Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 26061. 2 Department of Animal Sciences, University of Vermont, Bur- ruminants. Nonetheless, the fundamental aspects are lington, Vermont 05405. certainly similar between species. For example, a study 3 Department of Animal Nutrition and Physiology, Danish Institute with hypophysectomized-ovariectomized goats indiof Agricultural Sciences, Foulum, DK-8830 Tjele, Denmark. cated a critical role for GH in normal development 4 Gene Evaluation and Mapping Laboratory, USDA, ARS, Beltsville, Maryland 20705. (3). Moreover, goats and sheep, like rodents appear to 5 To whom correspondence should be addressed at: Department of depend on placental lactogen(s) to maximize mammo- Dairy Science, 2080 Litton Reaves Hall, Virginia Polytechnic genesis after conception (4,5). Institute and State University, Blacksburg, Virginia 24061-0315. Subsequent work has been focused on GH regulae-mail: rma@vt.edu tion of normal mammary development and the use of 6 Abbreviations: insulin-like growth factor one (IGF-I); insulin-like growth factor binding protein-1, 2 or 3 (IGFBP-1, 2, 3); growth GH to enhance mammary development. The availabil- hormone (GH); bovine somatotropin (bst). ity of recombinant bovine somatotropin (bst) has been 43 1083-3021/00/0100-0043$18.00/0 2000 Plenum Publishing Corporation

44 Akers et al. critical for these studies. Clearly, use of bst in lactat- (14). The mammary fat pad also increases in size, and ing dairy cows provides a potent husbandry tool for epithelial ducts penetrate this stromal tissue matrix. effective and safe stimulation of milk production (6,7). Increased estrogen and the presence of growth hormone Despite the widespread practical use of bst, the mechanisms induce the ducts to branch and elongate. During whereby milk production is increased are gestation, synchronous secretions of estrogen and progesterone poorly understood. Both bst and bovine placental (and placental hormones in goats and sheep) lactogen have been shown to enhance hormonally promote exponential mammary growth. Early gestation induced lactation in cattle (8,9). These effects likely is associated with final elongation and branching involve both proliferation and enhanced mammary of ducts, mid-gestation with formation of alveoli, and function. BST given during the nonlactating dry the later third of gestation with final proliferation and interval (10) between lactation periods in dairy cows differentiation of the secretory cells of the alveoli. might also increase subsequent milk production (11). Numerous studies have indicated that milk production Thus, these results suggest that bst enhances mam- is directly proportional to the number of mammary mary development and can be used as a management epithelial cells. Indeed, the correlation between total tool in dairy production. However, no discussion of mammary parenchymal DNA and milk production bst could be complete without careful consideration averages about 0.85 (4). of the insulin-like growth factor family of proteins. Histologically, the peripubertal rodent gland differs The focus of this paper is peripubertal development markedly from that of the ruminant. Rodent glands of the ruminant mammary gland, and specifically the exhibit widely scattered ducts with the entire structure relevance of the GH-IGF axis in this process. suspended in a sea of adipocytes interwoven with scat- tered blood vessels, fibroblasts, and leukocytes (15). Mammary development in ruminants is less well characterized. DUCTAL MORPHOGENESIS AND In fact, based on extensive histological study STROMAL INTERACTIONS SYSTEMIC of prepubertal lamb mammary glands (16), there is no VS. LOCAL REGULATION compelling evidence for presence of typical end buds. Figure 1A, shows a mid-sagittal section through one The epithelial cells, which give rise to the cells of mammary gland of a nine week-old ewe lamb. Examithe mammary alveoli, are derived from the ectoderm. nation of such sections and quantitative studies of These precursor cells first appear as a thickening of mammary glands of heifers and lambs (15,17) demonthe ventral epithelium of the body and proceed through strate that much of the mammary fat pad of prepubertal a developmental series described as the mammary ruminants is completely devoid of parenchymal tissue. band, streak, line, crest, hillock, and mammary bud. Cross-sectioned ducts from pre- and post-puberty In a heifer fetus, four mammary buds correspond to ruminants often exhibit a scalloped appearance, sugthe four glands of the udder. At birth the teat, a primary gesting a more complex tubular structure than in the sprout and several secondary sprouts are present rodent. Serial sections show abundant outpocketing of (12,13). Initially, the epithelial portion of the mammary epithelia along the course of the ductal tree, especially gland grows isometrically but at about three months when compared with the rodent. As gestation of age (prior to puberty) growth becomes allometric advances, clusters of alveolar structures appear as scat- Fig. 1. (opposite) Illustration of gross mammary development, histological appearance, and estrogen receptor staining of prepubertal ruminant mammary glands. Panel A shows an approximately mid-saggital section through one of the mammary glands of a 12-week-old ewe. The dark, dense mass to the left of the teat is the compact developing parenchymal tissue (PAR). A portion of the supramammary lymph node (arrow) appears in the surrounding stromal tissue toward the rear of the udder. Note how little of the mammary fat pad contains parenchyma and the compact nature of the parenchymal tissue. Unpublished photograph from Ellis and Akers (16). Panel B provides the histology of mammary parenchyma of a prepubertal heifer. Note the distinct compact arrangement of mammary ducts. The area indicated by the open box is similar to the region of tissue shown in panel C. The section is stained with H&E. Panel C shows a portion of parenchymal tissue from a slide processed to detect immunocytochemical staining for presence of estrogen receptor. Note the abundant number of darkly stained epithelial cell nuclei indicating the presence of the estrogen receptor. Some of these cells are indicated by arrows. A number of adjacent epithelial cells, often seemingly more closely associated with the lumen do not express the estrogen receptor. Expressing cells are also virtually absent in the surrounding stroma. The bar in the lower right of panel B and C is approximately 50 m. Images are from an unpublished study (Capuco, Akers, and Wood).

Local IGF-I Axis in Ruminants 45

46 Akers et al. tered, rounded islands. In late gestation, histological mechanism of action of GH remains a puzzle, but fields are filled with closely packed alveoli (18). In almost certainly involves the IGF family of related the ruminant, in contrast to rodents, the gland is not proteins, as well as locally produced factors, including filled with elongated ducts during the prepubertal receptors, binding proteins and perhaps other growth period waiting for subsequent development of side factors. branches. To use a plant analogy, in the peripubertal Despite the documented importance of estrogen rodent, widely spaced mammary ducts fill the mam- for mammary growth at puberty, there have been no mary fat pad like the bare branches of a tree. In the published attempts to use estrogen to stimulate mam- peripubertal ruminant closely packed ducts radiate mary growth in heifers in this period. Ovariectomy at from the gland cistern in broccoli-like fashion. The 2.5 months of age in Holstein heifers reduces mam- ducts generally fill only a fraction of the mammary mary parenchyma 85 90% (26). Consistent with an stroma. Since many hypotheses related to mammary involvement of estrogen, there was also a small, but development and function have arisen from rodent significant, reduction in the circulating level of estradiol studies, it remains to be seen if these ideas apply in the ovariectomized heifers. The level of estrastudies, equally well to other species. diol in heifers, however, is very low, and it is questionable whether a ten-fold reduction of parenchymal tissue mass could be due to such a small reduction OVARIAN REGULATION OF MAMMARY in serum concentration ( 0.1 pg/ml). Woodward et DEVELOPMENT AND MAMMARY al. (27) observed that administration of estrogen to TISSUE PRODUCTION OF IGF-I AND heifers resulted in a tissue specific increase in DNA IGF-I BINDING PROTEINS synthesis in mammary ducts. These results strongly support the rodent paradigm of the need for both GH The prepubertal bovine mammary gland responds and estrogen for ductal proliferation. In contrast, to both GH and ovarian secretions. While effects of despite dogma touting the essential role of the ovary complete removal of endogenous GH on mammary (4), Ellis et al. (28) convincingly demonstrated that development in the bovine have not been reported, a allometric mammary growth in prepubertal sheep is positive effect of bst on udder growth in heifers has ovary-independent. Apparently, presumptive universal been reported (19,20). Although GH-treatment stimu- regulatory pathways are not even assured between lates mammary growth before puberty, the results do ruminants. Thus, assumptions (between species) of not convincingly show that the effect is translated into similar regulation of mammary development and function increased milk yield. GH-treatment during late pregnancy should be made cautiously. Hovey et al. (29) have seems to stimulate both mammary growth and recently reviewed the marked structural, biochemical, milk yield during lactation (10,21). The limited data on and growth factor differences between species with the effect of GH on mammary growth during lactation respect to interactions between the mammary parenchyma suggest that mammary growth is unaffected by GHtreatment and surrounding stromal tissue. Steroid priming in early lactation, but GH increases the of calves with estradiol and progesterone for 9 18 d amount of mammary parenchyma in mid-lactation. increased mammary weight by more than 40% (648 A direct effect of GH on the mammary gland vs. 453 g). These results (30) support the concept requires specific receptors. However, it has not been of a sequential development of responsiveness of the possible to show specific binding of GH to pubertal mammary gland to various hormones, including estradiol. ruminant mammary tissue using ligand-binding assays Although neither primary nor immortalized (22,23). These results are consistent with previous bovine mammary epithelial cells proliferate in studies utilizing tissues from pregnant or lactating response to estrogen (31), bovine and ovine, mammary cows or sheep (5). Interestingly, it has been possible explants are responsive (32,33). Moreover, immunocytochemical to show expression of a 4.5 to 4.7 kb transcript of the staining indicates that bovine mammary GH receptor in mammary tissue (24,25). The existence epithelial cells strongly express estrogen receptor (Fig. of mrna for the GH receptor, however, does not 1B). Perhaps expression of the estrogen receptor necessarily mean that the receptor itself is found in requires the presence of the surrounding stromal cells. mammary tissue. The hypothesis that the effect of GH This finding would provide an explanation for the on mammary growth is indirectly mediated via IGF- failure of isolated epithelial cells to respond to estrogen. I has been the subject of increasing research. Thus the Thus, despite the fact that estrogen is of primary

Local IGF-I Axis in Ruminants 47 importance for mammary growth, its mechanism of action is not clear. Although the liver is the major source of circulating IGF-I, the physiological importance of locally synthesized IGF-I in the mammary gland is increasingly appreciated. Local IGF-I is stroma-derived in rodent, human and ruminant mammary glands (29). IGF-I expression was markedly higher in the mammary stroma than parenchyma, and increased during onset of allometric mammary growth in sheep (34). Differences between IGF-I expression in stromal and parenchymal tissue are difficult to evaluate because the epithelium in the developing udder is surrounded by stromal cells (see Fig. 1B). However, the finding of IGF-I expression in mammary stroma from intact and cleared mammary glands leaves no doubt that IGF-I synthesis occurs in the stroma (34). Detection of IGF-I transcripts in isolated stromal cells but not in acinar cells support this idea (35). Forsyth et al. (36) have recently provided in situ expression data for IGF-I, IGF-II, and IGF- I receptor in the fetal ovine mammary gland which convincingly confirm these conclusions. We have also recently reported effects of feeding level and bst treat- ment on expression of IGF-I and IGF binding protein transcripts in the developing heifer mammary gland (37). Northern blot analysis of mammary parenchymal tissue from prepubertal heifers showed a major 7.5- kb IGF-I mrna and a less abundant 9.5-kb mrna. There was a tendency for an interaction between bst treatment and feeding level on mammary expression of the 7.5-kb IGF-I transcript. Expression of mrna for IGFBP-3 (2.6 kb), IGFBP-2 (1.8 kb) and IGFBP- 1 (2.0 kb) was also detected in mammary tissue. High feeding level reduced levels of the IGFBP-1 mrna. In contrast, expression of IGFBP-3 and IGFBP-2 mrna was unchanged in mammary tissue by bst or feed- ing level. We (Akers et al., unpublished) have recently found that ovariectomy of prepubertal heifers at 4 months of age significantly inhibits mammary paren- chymal growth at 6 months of age. These data confirm the work of Purup et al., (26). We found that stromal IGF-I mrna was greater and IGFBP-3 mrna lower in control compared with ovariectomized heifers. These results support our hypothesis that mammary duct development in prepubertal heifers is modulated by local mammary tissue synthesis of IGF-I and IGFBP-3. EFFECT OF FEEDING LEVEL ON MAMMARY TISSUE DEVELOPMENT AND FUNCTION Rapid rearing of replacement dairy heifers to decrease the age at sexual maturity is advantageous because heifers can begin milk production at a younger age. However, data from many studies indicate that feeding level and especially high-energy intake can markedly impair mammary development and subse- quent milk production. Comprehensive reviews (38,39) suggest that during a critical interval prior to puberty an excessive rate of weight gain impairs mammary development and future milk production. The overall conclusion is that levels of feeding which support gains above 600 g/d in large dairy breeds cause a permanent reduction in future milk production. There is controversy as to the exact timing of this critical interval between breeds and questions of effects of diet composition but little doubt of its existence. A large Danish study (40) on the effect of prepubertal average daily weight gain on subsequent milk production in each of three dairy breeds reveals a consistent pattern of lost milk production as prepubertal weight gain increases. Because increased rates of gain can be achieved with a variety of diets, many workers have questioned whether negative effects of high weight gain during the prepubertal period might not be pre- vented by altered diet composition. Prepubertal heifers fed with three energy levels achieved calving ages of 24, 22, and 21 months (41). Milk yield (adjusted for body weight) was lower for heifers calving at 22 and 21 months (i.e., with higher rate of gain). However, protein source had no effect on subsequent milk yield. Mantysaari et al. (42) also found that protein source (urea vs. rapeseed meal) did not prevent a negative effect of rapid weight gain on udder growth (42). In a similar nutrition study (43), Holstein heifers were fed diets of alfalfa or corn silage for gains of 725 or 950 g/d. Eight heifers per treatment were necropsied for tissue evaluations. The results suggested a deleteri- ous effect of rapid prepubertal weight gain on mammogenesis and milk yield. Since it is known that bst levels in the blood are lower in rapidly growing heifers, several workers have examined the response of such heifers to bst supple- mentation. Radcliff et al. (44) evaluated the influence of elevated energy and protein in combination with daily injections of bst on growth and mammary devel- opment in 40 Holstein heifers. As expected, heifers fed for high gain weighed more and had more carcass

48 Akers et al. fat. However, feeding the high energy, high protein In recent studies (25,47) the relative importance diet did not reduce mammary parenchymal DNA. of IGF-I and IGFBP-3 in serum and mammary tissue Injections of bst increased mammary DNA by 47% extracts in stimulating mammary epithelial cell proliferation irrespective of diet. Subsequent milk yields nonetheless has been evaluated. Recombinant IGFBP-3 and were lower in animals previously given the high- IGF-I antibody were added to cultures of primary protein, high-energy diet in the absence of bst treatment. undifferentiated mammary epithelial cells supple- However, bst given in conjunction with a high mented with IGF-I, serum, or mammary gland extracts feed intake reduced age at first calving without reducing from prepubertal heifers. Addition of heifer serum milk yields (45). Thus, it may be possible to use stimulated cell proliferation at concentrations of 4% bst to negate some of the deleterious effects of and greater. Mammary extracts, on the other hand, overfeeding on mammary development. stimulated cell proliferation much more effectively Irrespective of dietary manipulation relative to than serum or IGF-I alone, yielding a mitogenic effects of rate of gain, mammary growth can be response more than twice that induced by the highest affected by specific components in the diet. Lambs IGF-I concentration tested. Maximal stimulation of fed protected polyunsaturated fat grew at a similar DNA synthesis occurred with a 6% concentration of rate to lambs fed a high energy concentrate diet, but mammary extract. To determine the contribution of mammary parenchymal mass and DNA were markedly IGF-I to mitogenic activity in serum and mammary increased at 22 weeks of age (17). Zhang et al. (46) extracts, IGFBP-3 and antibodies to IGF-I were added reported that 15 vs. 20% dietary protein did not affect to media containing either 50 ng/ml of IGF-I, 5% udder growth at six months of age. However in lactation serum or 5% mammary extracts. When added with ewes previously fed the high protein diet had more IGF-I, equimolar and greater concentrations of IGFBP- parenchymal mass, DNA, and tended toward higher 3 reduced DNA synthesis to the level of basal medium. milk yields. Such data serve to emphasize how little we The bioactivity of serum (5%) was reduced by 73 and understand of the complex interrelationships between 43% by mono and polyclonal antibodies at equimolar diet, peripubertal mammary development, and subse- concentrations, respectively, while recombinant quent milk production in dairy animals. IGFBP-3 completely abolished the effect of serum. An equimolar concentration of IGFBP-3 abrogated 35% of the mitogenic activity in extracts. However, the SOMATOTROPIN STIMULATION OF dose-dependent inhibition of IGFBP-3 was proportion- MAMMARY DEVELOPMENT AND ally greater at 2 (50% inhibition) and 4 times (82% INTERACTION WITH THE IGF-I AXIS inhibition) the molar concentrations of IGF-I present in mammary extract. In this study, the concentration How do effects of GH coordinate with steroid of IGF-I in the pooled mammary extract (33 ng/ml) regulation of peripubertal udder development and the was approximately one third of that in serum (107 ng/ IGF-I axis? A plethora of data show exogenous GH ml), yet the extract was nearly 20 times more mitogenic increases milk production in lactating ruminants. Simi- than serum. These data suggest that the IGFs indeed larly, GH treatment of heifers acutely stimulated mammary provide a considerable portion of the mitogenic activity epithelial cell proliferation (Berry, McFadden, available in serum and mammary tissue, either individ- and Akers, unpublished) and increased udder parenchymal ually or through interactions with other growth factors. mass over a period of days or weeks. Yet, there Purup et al. (24) have summarized data from the is little evidence that GH has a direct effect on the bioassay, IGF-I concentrations, and IGFBP profiles of mammary epithelium. Heifers or cows treated with individual serum samples, and mammary extracts for bst are consistently shown to exhibit increased circulating heifers in a 2 2 factorial experiment to test effects concentrations of IGF-I. High affinity IGF-I of feeding level and bst treatment. Sera were tested receptors are present in mammary epithelial explants, for capacity to stimulate mammary cell proliferation isolated bovine mammary cells are sensitive to IGF- to study relationships between IGF-I and IGFBP. DNA I, and mammary tissue synthesizes IGF-I (25,37). synthesis was positively correlated with concentrations Although the liver is believed to be the major source of IGF-I and IGFBP-3 but negatively with concentrations of circulating IGF-I, the physiological importance of of IGFBP-2. The concentration of IGF-I in locally synthesized IGF-I in the mammary gland extracts was much lower than for serum ( 30 vs. 200 should not be underestimated. ng/ml). However there was a strong positive correla-

Local IGF-I Axis in Ruminants 49 tion between serum and extract concentrations of IGF- secretion of recombinant ovine IGF-I (SV40-IGF-I I(r 0.84) suggesting at least some portion of the cells). A second cell line allows IGF-I secretion at a IGF-I in mammary extracts is likely serum derived. moderate rate (TK-IGF-I cells) and the third is constructed Unlike serum, there was no significant correlation with an inducible promoter (MD-IGF-I cells). between mitogenic activity of mammary extracts and Cultured on plastic, nontransfected parental cells IGF-I concentration. This may reflect the relatively respond to treatment with insulin, IGF-I, or IGF-II low concentration and narrow range for IGF-I in mam- with increased secretion of primarily IGFBP-2 (50,51). mary extracts (20 40 ng/ml) compared with serum However, TK- and SV40-IGF-I cells display a chronic (100 400 ng/ml). When compared relative to IGF-I increase in secretion of IGFBP-3 irrespective of treatment concentration, addition of 10% extract ( 3 ng/ml) was (49). Just as culture of MD-IGF-I cells on sixfold more stimulatory than addition of 3 ng/ml of Matrigel induces secretion of recombinant IGF-I (50), IGF-I. This result is surprising given that essentially incubation of control cells on collagen, laminin, fibroall of the mitogenic activity of mammary extract can nectin, or Matrigel induces a marked increased secretion be blocked with the addition of IGFBP-3. However, of IGFBP-3. Secretion of IGFBP is increased in a moderate negative correlation (r 0.51) between transgenic cells on these extracellular matrix materials mitogenic activity and IGFPB-3 content of extracts but there is little change in the profile of secreted suggests that local IGFBP-3 mediates response to binding proteins (Akers, unpublished data). Clearly, IGF-I. there is much to be learned about the interactions These data suggest that stimulation by serum and between changes in local synthesis/secretion of IGFmammary extracts depends on IGF-I. However, it is I, relevance of extracellular matrix, and impacts on very likely that locally produced IGFBP-3 inhibits ductal morphogenesis. Study of these cell lines offers mammary cell growth, either by associating with IGF- a potentially useful adjunct to experiments in intact I or perhaps by IGF-I independent actions. Relevance ruminants or with isolated mammary organoids alone. to the intact animal is indicated by the finding that There is hope that controlled modulation of local mammary tissue extract concentrations of IGFBP-3 secretion/synthesis of IGF-I in the mammary gland tend to be increased in high-fed animals (i.e., associated might lead to technologies to modify pubertal mam- with impaired mammary growth). mary development in dairy animals. We (52) recently reported precocious alveolar-bud formation in mammary glands of prepubertal transgenic mice in which IN VITRO MODELS FOR IGF-I MEDIATION recombinant IGF-I synthesis was directed to the mam- OF RUMINANT MAMMARY CELL mary gland by the mouse mammary tumor virus promoter. PROLIFERATION It is certainly a leap from mice to ruminants but such data suggest that the technologies being touted To model autocrine/paracrine secretion of IGF-I we have developed several lines of immortalized bovine mammary epithelial cells which secrete recombinant ovine IGF-I (48,49). Our objective is to learn how IGF-I signaling is modified by local secretion of IGF-I. A second goal is to understand how a model substratum (collagen) modulates IGF-I action and ductal morphogenesis. Our hypothesis is that IGFBP-3 is generally an inhibitor of IGF-I activity and is induced by secretion of IGF-I, but that complex interactions between the mammary cells, their substratum, and secreted extracellular molecules regulate sensitivity of the epithelium to IGF-I. Although it is possible to evaluate some effects of increased production of local IGF-I by addition of purified material to cultured cells, this approach only partially mimics paracrine and/or autocrine secretion. One of these cell lines contains a transgene, which induces a high rate of constitutive to use the udder as a bioreactor for human pharmaceuticals might also be harnessed to promote dairy interests as well. CONCLUSIONS There are approximately 9.3 million lactating dairy cows in the United States. Given that each cow averages three years in the herd, one third of these animals are replaced each year. Assuming an average replacement cost of $1,000 these animals represent an annual investment of 3.1 billion dollars. Is it reasonable to understand so little of the fundamental biology of early mammary development related to a national resource worth 3.1 billion dollars per year? Development of techniques to enhance mammary development might be reasonably expected to increase average herd

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