1.  INTRODUCTION

         Growth and development of the mammary glands in pubertal heifers is important for milk yield potential at maturity. The regulation of mammary growth involves complex interactions of many hormones and growth factors, of which some are growth-stimulating and others growth-inhibitory. Although the systemic importance of the hormones estrogen and growth hormone (GH), for regulation of growth of the mammary gland has been well documented, these hormones have no or very little stimulating effect on mammary epithelial cell growth in vitro (for review). Thus other factors of either systemic or local origin are of importance for growth and development of the mammary gland. In recent years, a number of growth factors and binding proteins have been discovered that may be involved in mediating the effects of estrogen and GH or have specific effects of their own. In the present paper, we present data from studies in which we have investigated the mitogenic effect of specific growth factors and binding proteins using an in vitro model of epithelial cells from 8-9 mo heifers. The factors studied include growth factors of the insulin-like growth factor (IGF) family, the epidermal growth factor (EGF) family, the fibroblast growth factor (FGF) family, the transforming growth factor-ß (TGF-ß) family, and two IGF binding proteins. Since factors are studied in the same cell culture system, it is possible to evaluate their relative importance in regulation of growth and development of the pubertal mammary gland.

2.  PRIMARY CELL CULTURES OF MAMMARY EPITHELIUM

      Mitogenic effects of growth factors have been investigated in different cell culture systems with mammary cells from different species at different developmental stages. Unfortunately, species specificity exists such that findings from laboratory animals cannot automatically be applied to ruminants. Therefore, comparisons of the observed growth-stimulatory or growth-inhibitory effects are difficult. To study the importance of growth factors for growth and development of the pubertal heifer mammary glands, mammary epithelial cells were isolated from heifers with body weights between 200 and 250 kg and 8-9 mo of age. At this body weight first estrus has normally not yet occurred and the heifers have significant amounts of mammary tissue showing allometric growth (it is growing at a faster rate than the body). Therefore heifers at this development stage are ideal for studying regulation of mammary growth.     
      Mammary epithelial cells were cultured in three-dimensional collagen gels as described previously. Briefly, tissue pieces excised aseptically form mammary parenchyma were digested in basal medium supplemented with collagenase, hyaluronidase, DNase and insulin. Organoids were isolated by filtration, centrifugation and precipitation, and subsequently stored in liquid nitrogen until use in cell culture experiments. Experimental designs always included 3 replicates per treatment. Cells were cultured for 24 h in basal serum-free medium 199 containing insulin (10 ng/ml), followed by an additional 4 d in treatment media containing growth factors and/or binding proteins. Culture media were changed every 2 d and 1 µCi [methyl-³H]thymidine was added for the last 24 h of the culture period.

DNA synthesis was measured as thymidine incorporation as described previously.


3.  INSULIN AND INSULIN-LIKE GROWTH FACTORS (IGFS)     

      Insulin and insulin-like growth factors are structurally homologues peptides that are involved in many biological processes. The biological effect of IGFs is influenced by association with IGF binding proteins (IGFBPs). IGFs and IGFBPs are detected in many biological fluids including serum, colostrum, milk, lymph and follicular fluid. IGFs and IGFBPs are mainly synthesized in the liver but also locally in many tissues including mammary tissue.In ruminants, the stimulatory effects of insulin, IGF-I, IGF-II and the natural variant of IGF-I, des-(1-3) IGF-I, on DNA synthesis in ruminant mammary tissue in cultures has been documented in a number of reports. These studies include undifferentiated mammary epithelial cells from 4-6 mo old heifers, mammary epithelial cells prepared from pregnant sheep and pregnant, nonlactating heifers, and prepartum and lactating cows. In undifferentiated mammary epithelial cells, des-( 1-3) IGF-I was found to be as potent as IGF-I, while IGF-II was significantly less active (Peri et al., 1992). In 8-9 mo old heifers, we found des-(1-3) IGF-I to be more potent than IGF-I, IGF-II and insulin (Fig. 1). Des-( 1-3) attained half-maximal activity at a concentration of less than 1.5 ng/ml, native IGF-I at 1.5-3 ng/ml, whereas IGF-II was required at 3-6 ng/ml before reaching half-maximal activity. However, the maximal proliferative activity of des-( 1-3) IGF-I, native IGF-I and IGF-II were not different, while insulin exhibited lower total activity and did not attain the maximal activity of the IGFs even in a concentration of 1000 ng/ml (this concentration not shown).


                                                
Fig. 1 Effect of IGF-I, IGF-II, des-(1-3) IGF-I and insulin on DNA synthesis, measured by incorporation of [methyl-³H]thymidine, into mammary epithelial cells prepared from 8-9 mo old prepubertal heifers and grown in 3-dimensional collagen gels. Values are LSmeans ± SEM of values obtained from cultures with triplicate samples. These results show that IGFs are very potent factors for mammary epithelial cell growth. Differences in potency of IGF-I, des-(1-3) IGF-I and IGF-II in different studies may depend on variation in affinity and number of IGF membrane receptors. Two types of IGF receptors (type 1 and 2) are known. We have confirmed the presence of type 1 IGF receptors in heifers at this developmental stage in ligand-binding assays with crude mammary membranes. The potency of the IGFs in different culture systems may also depend on the extent of IGF-binding protein (IGFBP) accumulation in the particular culture system since IGF bioactivity is modulated by these specific, high-affinity binding proteins. Mammary cells derived from pregnant, nonlactating heifers and grown in collagen gels show IGF-I inducible secretion of IGFBP-2 and IGFBP-3¹⁰. We have recently shown the abundance of mRNA and

protein for IGFBP-1, -2, -3 and -4 in mammary tissue from heifers suggesting the involvement of the binding proteins in regulating growth of mammary epithelial cells. We therefore studied the effect of recombinant IGFBP-2 at the cellular level by adding increasing concentrations to cell culture medium with (Fig. 2b) or without (Fig. 2a) IGF-I. IGFBP-2 alone did not have any significant (P>0.49) effect on DNA synthesis (Fig. 2a). However, when IGFBP-2 was added to cell culture medium containing IGF-I, IGFBP-2 abolished the bioactivity of IGF-I (Fig. 2b). Addition of IGFBP-3 to cell culture medium significantly inhibited the mitogenic effect of IGF-I and IGF-II but not des-(1-3) IGF-I (Fig. 3) in line with the reduced affinity of des-(1-3) IGF-I for IGF-binding protein In previous studies we found that IGFBP-3 alone also inhibited the mitogenic activity of serum and mammary tissue extracts from heifers at this stage of development.The evidence therefore suggests that locally produced IGFBP-3 may therefore inhibit mammary cell growth, either by associating with IGF-I or IGF-II and modulating its activity, or by an IGF-independent mechanism.

 4.   THE EPIDERMAL GROWTH FACTOR (EGF) FAMILY

     The epidermal growth factor family is comprised of at least ten proteins including epidermal growth factor (EGF), heparin binding EGF, transforming growth factor-α (TGF-α), amphiregulin and various heregulins.

      These proteins are a family of structurally related growth factors that act via the EGF (ErbB- 1) receptor, which is a specific high-affinity receptor located on the plasma membrane. Currently, little is known concerning the mammogenic role of EGF and related growth factors in ruminants. Despite the existence of specific EGF receptors in mammary tissue from sheep and cows, expression of EGF messenger RNA is so far known only from a single study with ruminant tissue. Expression of other proteins of the EGF family, however, occur in ruminants, as the bovine mammary gland expresses TGF-α. Recently, expression of mRNA for amphiregulin was shown in the sheep mammary gland. Amphiregulin, that was originally isolated from conditioned medium from MCF-7 human mammary carcinoma cell line has to our knowledge never been investigated in bovine mammary tissue.

     The mitogenic effect of growth factors of the EGF family has been examined in a limited number of studies with ruminant cells. EGF and TGF-α  both stimulated DNA synthesis in mammary tissue explants from midpregnant heifers, in mammary epithelial cells from pregnant heifers and cows and in alveolar cells prepared from pregnant sheep. DNA synthesis was also stimulated by EGF in undifferentiated mammary epithelial cells from 4-6 mo old heifers, by TGF-α in alveolar cells from non-pregnant, pregnant and lactating sheep, and by amphiregulin in alveolar cells prepared from pregnant sheep. Furthermore, EGF infused into the bovine mammary gland, caused an increase in DNA synthesis in the gland.

       To investigate the mitogen activity of EGF, TGF-α and amphiregulin in mammary tissue from prepubertal heifers, we tested their effect on DNA synthesis in undifferentiated mammary epithelial cells from 8-9 mo heifers. Proliferation of mammary epithelial cells was stimulated by EGF, TGF-α and amphiregulin but not as much as by growth factors from the IGF family (Fig. 4). Synthesis of DNA was increased about 3.5 and 1.75 fold over basal medium by EGF and TGF-α at a concentration of 50 ng/ml. The previous studies with alveolar cells from pregnant sheep also showed significant effects of both EGF and TGF-α but the effect of EGF was smaller and not clearly dose-related compared with TGF-α. Likewise, Collier et al. found that bovine mammary epithelial cells from pregnant heifers responded more to bovine TGF-α than to EGF. In the MAC-T bovine mammary epithelial cell line, EGF was reported to be without effect. The mitogenic effect of amphiregulin was significantly (P<0.01) increased in our study with a maximal effect (~30% increase) at a concentration of 100 ng/ml (Fig. 4) but the effect was smaller than the effect of EGF and TGF-α. The presence of mRNA for EGF, TGF-α and amphiregulin in the ruminant mammary gland and the results presented here, suggest that these growth factors may be important in regulation of mammary growth and development in prepubertal heifers. However, more information is needed about the suggested presence of these factors in mammary tissue.

5. THE FIBROBLAST GROWTH FACTOR (FGF) FAMILY

     The fibroblast growth factors (FGFs) are a family of heparin binding growth factors that consist of at least 15 structurally related polypeptide growth factors. The FGFs are widely distributed throughout the developing and adult body and display diverse biological activities, most of which are mediated via FGF tyrosine kinase receptors (FGFRs), of which five have been described until now. At least three of the FGFs are supposed to participate in the regulation of growth and function of mammary gland cells in ruminants. These are fibroblast growth factor-1 (FGF-l), also known as acidic FGF (aFGF), fibroblast growth factor-2 (FGF-2), also known as basic FGF (bFGF), and fibroblast growth factor-7 (FGF-7), also known as keratinocyte growth factor (KGF). Recently, it was shown that these FGFs and their receptors are expressed during development, lactation and involution in the bovine mammary gland. The highest mRNA concentrations of FGF-1, -2 and -7 were detected in the glands of virgin heifers and primigravid heifers during involution. Tissue concentrations of FGF-1 and -2 was also shown to be highest in these periods.

        The mitogenic effects of FGFs on mammary tissue has almost exclusively been studied in rodents. Using primary cell cultures, FGF-2 has been found to be growth-stimulatory for mouse mammary epithelial cells from all stages of mammary development. FGF-1 and -7 also stimulated growth of mammary epithelial cells from virgin mice. In ruminant mammary tissue, only one study exist in which the effect ofFGF-2 has been investigated. FGF-2 (0.1- 50 ng/ml) showed dose-dependent mitogenic activity in undifferentiated mammary epithelial cells from 4-6 mo old heifers. Using our primary mammary epithelial cell cultures, we have investigated the mitogenic effect of FGF-1 and -2. Fig. 5 shows that FGF-1 significantly increased DNA synthesis in concentrations above 0.5 ng/ml with maximum stimulation at the highest concentration (100 ng/ml) added to the cell culture medium. FGF-2, however, tended to increase D N Asynthesis at concentrations below 10 ng/ml, but significantly decreased D N Asynthesis when 50 and 100 ng/ml was added to culture medium. The stimulating effect ofFGF-1 is in agreement with previous studies in mice, while the mitogenic response to FGF-2 contrast the dose-dependent mitogenic activity in mammary epithelial cells from 4-6 mo old heifers. Anyhow, previous studies showing expression and tissue concentrations of FGFs and the present results suggest FGFs to be important in the local regulation of the bovine mammary gland.


6.  THE TRANSFORMING GROWTH FACTOR-ß (TGF-ß) FAMILY

      The TGF-ß family is composed of at least five related multifunctional proteins that can affect a variety of cellular functions such as extracellular matrix formation, differentiation and growth depending on the cell and tissue type . TGF-ß is involved in a variety of physiological and pathological processes, including wound healing in which TGF-ß produced in blood platelets induces accelerated healing of incisional wounds. Platelets represent the most concentrated natural source of TGF-ß1 (20 mg/kg), but TGF-ß1 is believed to be secreted from nearly all cells. Three isoforms of TGF-ß (TGF-ß1, TGF-ß2 and TGF-ß3) have been identified. The most well characterized member of the TGF-ß family is TGF-ß1, which in its biological active form is a 25 kDa disulphide-linked homodimer. TGF-ß1 is secreted in an inactive form bound to a 75 kDa glycoprotein known as the latency associated peptide (LAP). This latent TGF-ß1 complex has to be activated to obtain its biological functions. This activation may be important in controlling the physiological functions of TGF-ß1 in vivo.

      Very little is known about the role of TGF-ß in mammary development in ruminants. We have shown the existence of TGF-ß1 in prepubertal heifer serum in concentrations ranging from 7 to 30 ng/ml, and Plaut et al. has shown the existence of specific receptor binding to bovine mammary membranes from prepubertal and pubertal heifers as well as from lactating and dry cows. In addition, Plath et al showed expression of TGF-ß1 in the bovine mammary gland during mammogenesis, lactogenesis, galactopoiesis and involution. Both specific receptor binding and expression of TGF-ß1 mRNA was higher during the prepubertal and pubertal periods than during lactation. These data support a role of TGF-ß1 in regulation of pubertal mammary growth and development.

      The actions of TGF-ß1 are mediated by binding to highly specific cell surface receptors of which most cells have three types, designated type I, II and III. The type I and II receptors are supposed to be directly involved in signal transduction while the type III receptor has been postulated to enhance binding of TGF-ß1 to the signalling receptors. We have investigated the existence of the type I and II TGF-ß receptors in bovine mammary tissue from prepubertal heifers using immunohistochemistry. These studies showed extensive staining for both the type I and II receptor in the ductal epithelium making an effect of TGF-ß1, mediated through the receptor, possible in the heifer mammary glands (Purup et al., unpublished results).

        In accordance with this, we have investigated the biological effect of TGF-ß1 in primary cultures of mammary epithelial cells. A biphasic effect of TGF-ß1 was observed. Addition of 25 and 50 pg/ml of TGF-ß1 to cell culture medium stimulated DNA synthesis, while higher concentrations of TGF-ß1 (>100 pg/ml) inhibited DNA synthesis. Maximal inhibition occurred at 5 ng/ml, corresponding to a 66% inhibition of DNA synthesis. In addition, TGF-ß1 (5 ng/ml) inhibited 77 to 92% of the mitogenic effect of serum (5% added to cell culture medium) obtained from 24 prepubertal heifers, and adding increasing concentrations of TGF-ß antibody had a positive effect on mitogenic activity of serum.

                                      

Fig. 6 Effect of TGF-ß1 on DNA synthesis in primary cultures of mammary epithelial cells prepared from 8-9 mo prepubertal heifers and grown in 3-dimensional collagen gels. TGF-ß1 (12.5, 25, 50, 100, 250, 1000, 5000, 10000 pg/ml) was added to basal medium and DNA synthesis measured by incorporation of [methyL3H]thymidine in the last 24 h of the culture period. Values are LSmeans ± SEM. Data from Purup et al., 1999.

       To study the potency and relative importance of TGF-ß1 in regulation of pubertal mammary growth, we investigated whether TGF-ß1 can influence the mitogenic effects of growth factors of the IGF and EGF families. TGF-ß1 in different concentrations (Fig. 7) was added to cell culture medium containing IGF-I (25 ng/ml), IGF-II (50 ng/ml), des(1-3)IGF-I (50 ng/ml), INS (100 ng/ml), EGF (25 ng/ml), TGF-α (5 ng/ml) and amphiregulin (100 ng/ml). The maximal effect of TGF-ß1 was obtained with concentrations of 500 pg/ml. This concentration is 30 times lower than the observed mean concentrations in serum from prepubertal heifers. The inhibition of the mitogenic effects induced by the different growth factors at this concentration of TGF-ß1 corresponded to approximately 50% of the proliferation obtained without TGF-ß1 in the medium. It is interesting that this relative growth inhibition was of the same size for growth factors from both the IGF and EGF family, while the inhibition measured as dpm was significant higher for the growth factors in the IGF family than in the EGF family. These results show that TGF-ß1 is a very potent regulator of pubertal mammary growth in heifers.

7. OTHER GROWTH FACTORS

      A number of other growth factors, both stimulatory and inhibitory, have been described as factors that might play a role in regulating growth and development of mammary gland. Among these are mammary-derived growth factor-1 (MDGF- 1) and platelet-derived growth factor (PDGF). MDGF-1 has been detected in human and bovine milk as well as milk of other mammals. Bovine MDGF-1 is predominant in mammary gland secretions from pregnant heifers and in colostrum and MDGF- 1 from human milk was observed to be growth stimulatory in normal and immortalized human mammary epithelial cells and in MCF-7 cells. PDGF is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. PDGF is secreted by epithelial cell lines derived from normal human tissue and from a number of breast cancer cell lines. Receptors for PDGF, however, do not seem to be present at either breast cancer or normal mammary epithelial cells, suggesting the effect of PDGF on epithelial cells to be indirect via the stroma. Mammary-derived growth inhibitor (MDGI), a member of a family of fatty acid-binding proteins, has been isolated from mammary tissue of pregnant and lactating ewes as well as the lactating bovine mammary gland. MDGI is expressed in lobulo-alveolar and ductal cells of pregnant and lactating animals but not in mammary epithelial cells of virgin animals. The physiological role of MDGI remains speculative. The postulated growth inhibitory function of MDGI is based on studies with transformed cells but so far no inhibitory effect on normal mammary epithelial cells in vivo or in primary culture has been demonstrated. Its role in the mammary gland appears to be as a differentiation factor rather than a an anti-proliferative factor.

8.  SUMMARYAND CONCLUSIONS