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The Expression of the IGF Family During Mouse Mammary Gland Development
Available online at w.sciencedirect.com
Agricultural Sciences in China
2007, 6(9): 1149-1156
ScienceDirect
September 2007
The Expressionof the IGF Family During Mouse Mammary Gland Development ZHAO Feng and LI Qing-zhang The Key Laboratory of Dairy Science, Ministry of Educution/Northeast Agricultural University, Harbin 150030, P . R. China
Abstract This study was to determine the patterns and levels of IGF family members' expression during postnatal mammary gland development. The authors investigated the protein expression profile of the major components of the IGF axis in murine mammary glands. All the proteins examined, IGF- I , IGF- 11, and IGF- 1 receptor (IGF- 1 R) were expressed at greatly different levels and displayed unique expression profiles. IGF- 11 and IGF- I R were always expressed at significantly higher levels than IGF- I . IGF- I was localized in adipocytes as well as the epithelial and stromal compartments, but just distinctly expressed where mammary cells aggregated to form ducts, in virgins. The IGF- 11 was localized only on the basal layer epithelial cell membranes of ducts and alveoli, with a peak level on the initiation of lactation. The higher level of IGF- I R compared with IGF- I was also found in adipocytes as well as in the epithelial and stromal compartments, especially during pregnancy and late lactation. The IGF- I R pathway was obviously significant for the development of the mammary parenchyma and stroma. Overall, the comparison of the expression profiles of these different proteins would strongly suggest that they were likely to have different functions throughout the mammary gland development, and it also highlighted the potential interactions and coregulation of the members of this axis. It seems that IGF- 11 was the major local modulator rather than IGF- I by an IGF- I R-independent pathway, especially for initiation of lactation. This study has demonstrated the importance and complexity of the IGF axis during mammary gland development and provides a valuable resource for future research in this area.
Key words: mammary gland, insulin-like growth factors, receptor, laser scanning confocal microscope (LSCM)
INTRODUCTION The insulin-like growth factor (IGF) system includes insulin-like growth factors (IGF- I ,IGF- I1 ), insulinlike growth factor receptors (IGF- I R, IGF- I1 R), and insulin-like growth factor binding proteins (IGFBPs). This IGF axis is important for the regulation of mammary development (Pacher et al. 2007). IGFs may act as mediators for some hormones, to control mammalian cell proliferation, differentiation, and apoptosis during mammary gland development. IGF- I can act in a paracrine or endocrine manner. It is believed to mediate
galactopoietic effects from exogenous bovine growth hormone (GH) in the cow (Ilkbahar et al. 1999; Tucker 2000). IGF- I mRNA is detected in stromal cells in the bovine mammary gland, but not in the epithelial cells (Baumrucker and Erondu 2000). However, IGF- I mRNA is positive in both types of cells in mice (Boutinaud et al. 2004). The liver is supposed to be the primary source of circulating IGF- I , which may affect target tissues such as the mammary gland. Most of the biological actions of IGF- I are mediated by the IGF- I receptor (IGF- I R), a membrane-bound heterotetramer with potent anti-apoptotic and cell survival activities (Hadsell 2003; Glait et al. 2006). It
Received 29 January, 2007 Accepted 11 May, 2007 ZHAO Feng, Ph D, E-mail: erjinzhi" 126.com; Correspondence LI Qing-zhang, Professor,E-mail: [email protected]
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binds IGF- I with a higher affinity than IGF- I1 or insulin. Different IGF- I R mRNA transcripts have been detected in the mammary gland. IGF- I1 is generally implicated as a locally derived growth factor that functions in embryonic tissues (Nielsen 1992). However, several lines of evidence support the role of IGF- I1 during normal postnatal mammary gland development. IGFI1 mRNA is heterogeneously expressed by MEC in the ductal epithelium of nulliparous mice (Richert and Wood 1999). Transgenic overexpression of IGF- I1 in the mammary glands of mice, from the ovine P-lactoglobulin promoter, results in epithelial hyperplasia (Bates et al. 1995), whereas, overexpression of IGF- I1 from the moloney murine leukemia virus promoter results in delayed involution and apoptosis of mammary epithelial cell (MEC) and the sustained expression of Akt (Moorehead et al. 2001). The insulin-like growth factor- I1 receptor (IGF- I1 R) regulates the level or activity of numerous proteins, including the factors that control growth and differentiation. IGF- I1 R is more specific for IGF- I1 than for IGF- I and does not bind insulin. Although IGF- I1 R (the mannose-6-phosphate receptor) represents an exclusive receptor for IGF- 11, it targets IGF- I1 for lysosomal degradation without evoking a cellular response (Wise and Pravtcheva 2006). In addition, IGF- I R has generally been considered as the main pathway for IGF- I1 action, because IGF- I1 displays a low affinity for insulin receptor (IR) (Nielsen 1992). Indeed, IGF- I R is expressed in the normal mouse mammary gland during development (Richert and Wood 1999). However, several disparate results arising from mouse genetic models for IR, IGF- I R, and IGF- I1 have continued to imply a role for the IR in the IGF- I1 action. Recenty, some researches have displayed that IGF- I1 functions as an autocrine/ paracrine effector of prolactin (PRL) action in the normal mouse mammary gland. They have found that PRL induces transcription of IGF- I1 in MEC via a prolactin receptor (PRLR)-dependent pathway that leads to the stimulation of alveolar development. They have also found that PRL induces IRS-1 and IRS-2 in the mouse mammary gland, indicating a role for IGF- I1 as a local effector of PRL action during mammary gland development (Hovey et al. 2003). Published data point toward the role of the IGF
system during the developmental or apoptotic processes in the mammary gland. However, very little is known about their expression of protein level in the different stages. The goal of this descriptive study is to examine the expression pattern of IGF- I and - I1 and IGF- I R in the defined stages of mammary gland growth and lactation in mice. All the data should help in more experimentally oriented future studies, to discover the possible role of the IGF system in the mammary gland.
MATERIALS AND METHODS Animals, tissue samplings, and preparation For the pregnancy ages, adult Kunming female virgin mice were placed in mating and checked daily for vaginal plugs. Additional pregnant mice were allowed to deliver their pups for analyses of glands in lactation and involution. The morning when the vaginal plug appeared was considered as day 0. For lactation and involution studies, litters were culled or pups were cross-fostered on day 1, to normalize to eight pups per dam. In all studies, the number four abdominal glands were removed within 20 min after slaughtered in defined stages, and small pieces (1-2 g) of mammary tissue were flash frozen in liquid nitrogen for cryostat sectioning, and for immunofluorescence studies. At least three glands from individual mouse were analyzed for protein expression. Frozen cryostat sections (8-10 pm) were mounted onto slides coated with APES and stored at 4°C until use for immunofluorescence. The classification of the animals was established as follows: (1) virgin (D20, D30, D40, D50, D60, n = 3), (2) pregnancy (PO, P6, P12, P18, n=3), (3) lactation (Ll, L4, L8, L12, L16, L20, L24, L28, L32, n=3), (4) forced involution (remove litters from lactating mice at five-day lactation, L6&11, L7&12, L8&13, L9&14, n = 3).
lmmunofluorescence The presence of IGFs and IGF- I R was demonstrated by using the indirect immunofluorescence technique. Nonspecific protein binding was eliminated by incubation with 10% normal goat serum in PBS for 1 h at room
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The Expression of the IGF Family During Mouse Mammary Gland Development
temperature. Sections were then incubated overnight at 4°C with a goat polyclonal antibody to IGF- I (dilution 1:lOO) or a rabbit polyclonal antibody to IGF- 11 and IGF- I R (dilution 1:100), and for 1 h with FITCconjugated rabbit anti-goadgoat anti-rabbit IgG (diluted 1:200 in PBS-1% BSA). Between each step, sections were washed thrice in PBS. All incubations were carried out in humidified chambers to prevent evaporation. Sections were lightly counterstained in PI for 5 min at room temperature, diluted to I pg mL' in PBS. Controls were performed by replacing the primary antibody with normal IgG at the same or greater concentration.
Statistical analysis When the data subjected to one-way ANOVA showed significant differences, a Bonferroni test was performed.
RESULTS Mammogenesis (mammogenesis in virgin mouse) Mammogenesis in nonpregnant mice was mainly characterized by ductal growth. The mammary gland was characterized by a well-developed adipose tissue in which small groups of ducts could be seen. The condensed connective tissue surrounded the ductular epithelium. Between the ducts, areas of loose connective tissue were seen, which contained comparatively few cells. As shown in Fig. 1-A the interlobular stroma and the epithelium, where mammary cells aggregated, appeared positive for immunoreactive IGF- I . Many of the adipocytes also showed a positive IGF- I staining in their cytoplasm. As shown in Fig. 1-E, only the basal layer of the ductal epithelium was with strong immunostaining for IGF- I , whereas, the luminal layer of the ductal epithelium, the interlobular stroma, and adipocytes were negative or displayed only weak immunostaining for IGF- 11. As shown in Fig. 1-I, all compartments were positive for immunoreactive IGF- I R, but it was obviously stronger in the adipocytes than in the epithelium surrounding those cells along their cell membrane.
Alveolargenesis (days 1-18 of pregnancy of primigravid mouse) At the beginning of this period, the alveoli were well developed to lobuli. The alveoli were small, roundish and lined by a cuboidal epithelium, which appeared negative for immunoreactive IGF- I . The adipose tissue was well developed at the beginning and displayed a positive IGF- I staining. Later in this period, the number of adipocytes was reduced. The glandular epithelium was always negative for IGF- I . In the stroma, no significant changes were seen. Adipocytes were generally IGF- I positive in their cytoplasm. In late pregnancy, the amount of adipose tissue around the lobuli was further reduced, but the remaining adipocytes still appeared immunoreactive for IGF- I . No immunostaining for IGF- I was seen in the secretory epithelial cells. The alveolar epithelial cells appeared with a distinct positive staining for IGF- 11, and as shown in Fig.1-F, only the basal layer epithelium of the alveoli displayed IGF- I1 immunoreactive in early pregnancy. The alveoli in late pregnancy were still strongly positive for IGF- 11, but it was difficult to determine whether the positive staining was only in the basal layer epithelium. As shown in Fig.1-J, all compartments remained positive for immunoreactive IGF- I R with striking enhancement, especially in the epithelium of the acini.
Lactation (days 1-20of post-partum) The lobuli of the mammary gland consisted of numerous alveoli with wide lumina, separated by small amounts of connective tissue. Within the alveoli, a large amount of secretion occurred. As shown in Fig.1-C, the secretory epithelial cells showed a negative or weak staining for immunoreactive IGF- I . The interlobular stroma displayed weak immunonegative staining for IGF- I . The alveolar epithelial cells appeared with a distinct positive staining for IGF- 11 as shown in late pregnancy, with a distinctly strong staining on day four of lactation (Fig. 1-G). The positive staining for IGFI R was more stnking in lactation than any other period all round the whole slice, with an extremely strong staining on day 16 of lactation.
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Natural involution (days 21-32 of post-partum) After the mouse had lactated for 20 days, the appearance of the mammary parenchyma changed significantly. The alveoli were irregular in their outline and were lined by a secretory epithelium. Lobuli with small and roundish acini were distributed between areas, with large acini having irregular outlines and a folded epithelium. As in the previous stages, little IGF- I immunoreactivity was seen in the secretory cells of any of the acini or ducts. The number of weak IGFI -positive fat cells in the stroma was low. The density of the intralobular stroma had increased considerably and it stained weakly for IGF- I (Fig.1-D). A weak to distinct IGF- I1 immunoreactivity occurred in the alveoli, and in particular those depauperated alveoli were easily discerned by their pronounced staining for IGF11 (Fig. 1-H). IGF- I R positive staining remained distinct in the epithelium and the stroma, but predominantly in the adipocytes that reoccurred (Fig. 1-L).
Forced involution (24-96 h after forced weaning) 0-96 hours after weaning, since day 5 of lactation, the appearance of the mammary structure changed significantly as seen in the natural involuting process, but by a flash pattern. Ninety-six hours after weaning, the original alveolar structural of the mammary gland had completely regressed. The mammary gland completed a recombination on the whole. As in the previous stages, weak IGF- I immunoreactivity was seen in the mammary gland. Only in the fat tissue and the stroma, a few cells were weakly IGF- I -positive (Fig.2-A-D). A distinct to weak to distinct IGF- I1 immunoreactivity occurred in the alveoli, and in particular those depauperated alveoli were easily discerned by their pronounced staining for IGF- I1 (Fig. 2-E-H). IGF- I R positive staining remained distinct in the epithelium and the stroma, and it also had a distinct to weak to distinct pattern. 0-96 hours after weaning, IGF- I R was localized predominantly in the epithelium and the stroma. At 72 hours, IGF- I R-positive staining was obviously weak on the whole slice. At 96 hours, IGF- I R-positive staining was distinct again and predominant in the reoccurred adipocytes (Fig.2-I-L).
ZHAO F e w et al.
Quantified analysis of protein levels of IGF family members’ expression in different development stages The patterns of IGFs and IGF- I R expression were examined by semi-quantified densitometric analysis of five pieces of corresponding laser scanning confocal microscope (LSCM) pictures, one of each of the three parallel groups. IGF- I slightly expressed at most stages, but was relatively stronger in the virgin. The highest IGF- I level was found in the mammary tissue of the day 50 virgin. The expression decreased significantly during lactation, followed by a slight increase in late lactation and involution (Fig.3-A and -D). The relative IGF- I expression of the virgin mice was obviously lower when compared to the IGF- I1 analysis. As shown in Fig.3-B and -D, IGF- I1 was strongly expressed in most stages, especially during middle pregnancy and early lactation. The highest IGFI1 level was found in the mammary tissue of day 4 lactation. Relative expression of IGF- I1 showed very significant differences in mammogenesis, pregnancy, lactation, and involution, and decreased significantly in early involution ( P <0.05). As shown in Fig.3-C and -E, the higher level of IGFI R expression was detected in late lactation (a clear increase appeared on day 16 of lactation) and the lower level in mammogenesis and early lactation. On the whole, the relative IGF- I R expression was obviously higher when compared with the other two analyses.
DISCUSSlON In general, the low abundance of IGF- I in almost all stages seems to suggest a minor function of the local IGF- I in the mouse mammary gland development, than endocrine IGF- I . It may be involved in duct morphogenesis and duct branching by a paracrine/ autocrine pattern in virgin mouse mammary gland. Local IGF- I , produced by mammary gland expresses both in the parenchyma and the stroma, showing its multifunctions. Epithelial-specific loss of IGF- I , during pubertal growth, results in deficits in ductal branching. In contrast, heterozygous reduction of IGF- I throughout the gland decreases expression of cyclins A2 and B1 during pubertal growth, and results in
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Fig. 1 Immunofluorescence analysis of IGF- I ,IGF- 11, and IGF- I R during normal mouse mammary gland development. PI counterstain. Magnification, 400 x. A-D, IGF- I in mouse mammary gland, D50, P6, L16, L28; E-H, IGF- TI in mouse mammary gland, D50, P6, L4, L28; I-L, IGF- I R in mouse mammary gland, D40,P6, L16, L28. Scale bars are about 50 pm.
Fig. 2 Immunofluorescence analysis of of IGF- I , IGF- 11, and IGF- I R in forced involuting mouse mammary gland. PI counterstain. Magnification, 400 x. A-D, IGF- I in mouse mammary gland, L6&11, L7&12, L8&13, L9&14; E-H, IGF- I1 in mouse mammary gland, L6&11, L78rI2, L8&13, L9&14; I-L, IGF- I R in mouse mammary gland, L6&11, L7&12, L8&13, and L9&14. Scale bars are about 50 pm.
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Fig. 3 Densitometric analysis of IGFs and IGF- I R in mouse mammary gland. Data from densitometric analysis are shown as means f SD (n=3). Significant differences (P<0.05) are indicated for IGF- I ,IGF- 11. and IGF- I R.
alterations in proliferation of the alveolar epithelium and milk protein levels during pregnancy-induced differentiation. Reduction in epithelial IGF- I at either of these stages has no effect on these indices (Richards et al. 2004; Loladze et al. 2006). Interestingly, a higher expression where mammary cells aggregate to form ducts indicates its actions involved in ductal morphogenesis in virgin mouse (Ruan and Kleinberg 1999; Ruan et al. 2005). The expression of IGF- I in adipocytes implies a potential effect of IGF- I on the proliferation and differentiation of adipocytes in this stage. The low level of IGF- I in pregnancy and lactation suggests that it is endocrine IGF- I which
regulates the mammary development in pregnancy and maintains milk production in lactation, and not local IGF- I . Both IGF- I and IGF- I1 have some similar functions for regulating mammary development alternatively or corporately. Similar to IGF- I , IGFI1 can also stimulate cell proliferation and be a survival factor of mammary epithelial cells in vitro, but with less potency than IGF- I (Pacher et al. 2007). IGFI1 mRNA has been detected in the mammary epithelial cells of both ruminants and rodents. The data further display that IGF- I1 level is obviously higher than IGFI in each mammary development stage, and increases
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The Expression of the IGF Family During Mouse Mammary Gland Development
dramatically in pregnancy and early lactation when compared to the constant low level in involution. These results indicate that locally produced IGF- I1 may play a greater role than locally produced IGF- I , with a potential positive action on alveolar morphogenesis and initiation of lactation. Hovey et al. (2003) had demonstrated that PRL enhances IGF- I1 mRNA expression, and IGF- I1 acted as the mediator of PRL in the mouse mammary gland during duct branching and alveolar development. From those data it is found that IGF- I1 may also act as a mediator of PRL on the onset of lactation, when PRL expression level is greatly upregulated, to initiate lactation (Tucker 2000; Freeman et al. 2000; Iwasaka et al. 2000). IGF- I1 is specially expressed in the basal layer epithelial cells of ducts and alveoli. It suggests that by a certain receptor or in an unknown manner, IGF- I1 is arrested in the basal layer epithelial cells membrane. It is impossible to answer for IGF- I R, because of positive staining for IGF- I R surrounding the cells along the membrane. It implies there is an IGF- I R-independent pathway for IGFI1 as is proved in pregnancy. As a survival factor of mammary epithelial cells, the decrease of IGF- I1 expression in involution following the weaning (natural or forced) may be essential to allow mammary cells apoptosis. The expression of IGF- I R is distinct in all stages examined, especially in early pregnancy (day 6 of pregnancy), and later lactation (day 16 of lactation). These results display that IGF- I R is still a vital receptor of IGFs during mammary gland development, even though there is a low abundance of local IGF- I and possible IGF- I R and independent action of IGF11. Some studies of transgenic mice that overexpress IGF- I during pregnancy and lactation h a v e demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation, but has minimal effect in early lactation on milk composition or milk production (Hadsell et al. 2002). These results show a distinct increase of IGF- I R in late lactation compared with those in the previously examined stages. It leads to the conclusion that IGF- I R-dependent pathways are involved in the maintenance of milk production in late lactation. This may be a direct effect of IGFs or of synergistic actions with other growth factors. In
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contrast, the low level of local IGF- I and the high expression of IGF- I R may suggest an increased importance of the endocrine IGF- I (produced by the liver), and may explain the well-known galactopoietic effect of exogenous GH. On the other hand, overactivation of IGF- I R in late lactation and within 48 h after weaning may result in loss of apico-basal polarity in mammary epithelial cells for involution as early lesions of breast cancer (Yanochko and Eckhart 2006). IGFI R-positive staining is distinctly localized in adipocytes and stromal cells, as well as the epithelial cells of ducts and acini. It indicates the significance of the IGF- I R-dependent pathway for the regulation of fat tissue and the stroma, in addition to the epithelium, during mammary gland development. In conclusion, the locally produced IGF- I and IGFI1 play a key role during mouse mammary gland development. IGF- I acts on the gland by IGF- I R. With a potential major function, in contrast to the former, the latter may act as a mediator of PRL action by an IGF- I R-independent pathway during lactation, which is the same as in mammogenesis and pregnancy. Given the high level of IGF- I R and the low abundance of local IGF- I in lactation, the action of endocrine IGF- I should be valued (Plath-Gabler et al. 2001). The increase of IGF- I R and the decrease of IGF- I1 following weaning can be important for the initiation of involution.
Acknowledgements This study was supported by Natural Science Foundation of Heilongjiang Province, China (ZTN0504).
References Bates P, Fisher R, Ward A, Richardson L, Hill D J, Graham C F. 1995. Mammary cancer in transgenic mice expressing insulinlike growth factor 11 (IGF- 11 ). British Journal of Cancer, 72, 1189-1193. Baumrucker C R, Erondu N E. 2000. Insulin-like growth factor (IGF) system in the bovine mammary gland and milk. Journal of Mammary Gland Biology and Neoplasia, 5,5344. Boutinaud M, Shand J H, Park M A, Phillips K, Beattie J, Flint D J, Allan G J. 2004. A quantitative RT-PCR study of the mRNA expression profile of the IGF axis during mammary gland development. Journal of Molecular Endocrinology, 33, 195-207. Freeman M E, Kanyicska B, Lerant A, Nagy G. 2000. Prolactin:
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structure, function, and regulation of secretion. Physiological Reviews, 80, 1523-1631. Glait C, Tencer L, Ravid D, Sarfstein R, Liscovitch M, Werner H. 2006. Caveolin-1 up-regulates IGF- I receptor gene transcription in breast cancer cells via S p l - and p53dependent pathways. Experimental Cell Research, 312,38993908. Hadsell D L, Bonnette S G, Lee A V. 2002. Genetic manipulation of the IGF- I axis to regulate mammary gland development and function. Journal of Dairy Science, 85,365-377. Hadsell D L. 2003. The insulin-like growth factor system in normal mammary gland function. Breast Disease, 17,3-14. Hovey R C, Harris J, Hadsell D L, Lee A V, Ormandy C J, Vonderhaar B K. 2003. Local insulin-like growth factor- 11 mediates prolactin-induced mammary gland development. Molecular Endocrinology, 17,460-471. Ilkbahar Y N, Thordarson G, Camarillo I G, Talamantes F. 1999. Differential expression of the growth hormone receptor and growth hormone-binding protein in epithelia and stroma of the mouse mammary gland at various physiological stages. Journal of Endocrinology, 161,77-87. Iwasaka T, Umemura S, Kakimoto K, Koizumi H, Osamura Y R. 2000. Expression of prolactin mRNA in rat mammary gland during pregnancy and lactation. Journal of Histochemistry and Cytochemistry, 48,389-395. Loladze A V, Stull M A, Rowzee A M, DeMarco J, Lantry 111 J H, Rosen C J, LeRoith D, Wagner K-U, Hennighausen L, Wood T L. 2006. Epithelial-specific and stage-specific functions of insulin-like growth factor- I during postnatal mammary development. Endocrinology, 147,5412-5423. Moorehead R A, Fata J E, Johnson M B, Khokha R. 2001. Inhibition of mammary epithelial apoptosis and sustained phosphorylation of Akt/PKB in MMTV-IGF- 11 transgenic mice. Cell Death and DijJerentiation, 8, 16-29. Nielsen F C . 1992. The molecular and cellular biology of insulinlike growth factor 11. Progress in Growth Factor Research, 4, 257-290.
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Pacher M, Seewald M J, Mikula M, OeNer S, Mogg M, Vinatzer U, Eger A, Schweifer N, Varecka R, Sommergruber W, Mikulits W, Schreiber M. 2007. Impact of constitutive IGFlf IGF2 stimulation on the transcriptional program of human breast cancer cells. Carcinogenesis, 28,49-59. Plath-Gabler A, Gabler C, Sinowatz F, Berisha B, Schams D. 2001. The expression of the IGF family and GH receptor in the bovine mammary gland. Journal ofEndocrinology, 168, 39-48. Richards R G, Klotz D M, Walker M P, Diaugustine R P. 2004. Mammary gland branching morphogenesis is diminished in mice with a deficiency of insulin-like growth factor- I (IGFI ), but not in mice with a liver-specific deletion of IGF- I . Endocrinology, 145,3106-31 10. Richert M M, Wood T L. 1999. The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions. Endocrinology, 140, 454-46 1. Ruan W F, Kleinberg D L. 1999. Insulin-like growth factor I is essential for terminal end bud formation and ductal morphogenesis during mammary development. Endocrinology, 140,5075-5081. Ruan W F, Monaco M E, Kleinberg D L. 2005. Progesterone stimulates mammary gland ductal morphogenesis by synergizing with and enhancing insulin-like growth factorI action. Endocrinology, 146, 1170-1178. Tucker H A. 2000. Symposium: Hormonal regulation of milk synthesis: hormones, mammary growth, and lactation: a 41year perspective. Journal of Dairy Science, 83, 874-884. Wise T L, Pravtcheva D D. 2006. Delayed onset of Zfl-induced mammary tumors in Igf2r transgenic mice. Cancer Research, 66, 1327-1336. Yanochko G M, Eckhart W. 2006. Type I insulin-like growth factor receptor over-expression induces proliferation and antiapoptotic signaling in a three-dimensional culture model of breast epithelial cells. Breast Cancer Research, 8, R18. (Edited by WANG Lu-han)
Q2007, C MS. All tights resewed. Publishedby Elsevierltd.
Agricultural Sciences in China
2007, 6(9): 1149-1156
ScienceDirect
September 2007
The Expressionof the IGF Family During Mouse Mammary Gland Development ZHAO Feng and LI Qing-zhang The Key Laboratory of Dairy Science, Ministry of Educution/Northeast Agricultural University, Harbin 150030, P . R. China
Abstract This study was to determine the patterns and levels of IGF family members' expression during postnatal mammary gland development. The authors investigated the protein expression profile of the major components of the IGF axis in murine mammary glands. All the proteins examined, IGF- I , IGF- 11, and IGF- 1 receptor (IGF- 1 R) were expressed at greatly different levels and displayed unique expression profiles. IGF- 11 and IGF- I R were always expressed at significantly higher levels than IGF- I . IGF- I was localized in adipocytes as well as the epithelial and stromal compartments, but just distinctly expressed where mammary cells aggregated to form ducts, in virgins. The IGF- 11 was localized only on the basal layer epithelial cell membranes of ducts and alveoli, with a peak level on the initiation of lactation. The higher level of IGF- I R compared with IGF- I was also found in adipocytes as well as in the epithelial and stromal compartments, especially during pregnancy and late lactation. The IGF- I R pathway was obviously significant for the development of the mammary parenchyma and stroma. Overall, the comparison of the expression profiles of these different proteins would strongly suggest that they were likely to have different functions throughout the mammary gland development, and it also highlighted the potential interactions and coregulation of the members of this axis. It seems that IGF- 11 was the major local modulator rather than IGF- I by an IGF- I R-independent pathway, especially for initiation of lactation. This study has demonstrated the importance and complexity of the IGF axis during mammary gland development and provides a valuable resource for future research in this area.
Key words: mammary gland, insulin-like growth factors, receptor, laser scanning confocal microscope (LSCM)
INTRODUCTION The insulin-like growth factor (IGF) system includes insulin-like growth factors (IGF- I ,IGF- I1 ), insulinlike growth factor receptors (IGF- I R, IGF- I1 R), and insulin-like growth factor binding proteins (IGFBPs). This IGF axis is important for the regulation of mammary development (Pacher et al. 2007). IGFs may act as mediators for some hormones, to control mammalian cell proliferation, differentiation, and apoptosis during mammary gland development. IGF- I can act in a paracrine or endocrine manner. It is believed to mediate
galactopoietic effects from exogenous bovine growth hormone (GH) in the cow (Ilkbahar et al. 1999; Tucker 2000). IGF- I mRNA is detected in stromal cells in the bovine mammary gland, but not in the epithelial cells (Baumrucker and Erondu 2000). However, IGF- I mRNA is positive in both types of cells in mice (Boutinaud et al. 2004). The liver is supposed to be the primary source of circulating IGF- I , which may affect target tissues such as the mammary gland. Most of the biological actions of IGF- I are mediated by the IGF- I receptor (IGF- I R), a membrane-bound heterotetramer with potent anti-apoptotic and cell survival activities (Hadsell 2003; Glait et al. 2006). It
Received 29 January, 2007 Accepted 11 May, 2007 ZHAO Feng, Ph D, E-mail: erjinzhi" 126.com; Correspondence LI Qing-zhang, Professor,E-mail: [email protected]
02007,CAAS. All rights reserved. Published by Elsevier Ltd.
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binds IGF- I with a higher affinity than IGF- I1 or insulin. Different IGF- I R mRNA transcripts have been detected in the mammary gland. IGF- I1 is generally implicated as a locally derived growth factor that functions in embryonic tissues (Nielsen 1992). However, several lines of evidence support the role of IGF- I1 during normal postnatal mammary gland development. IGFI1 mRNA is heterogeneously expressed by MEC in the ductal epithelium of nulliparous mice (Richert and Wood 1999). Transgenic overexpression of IGF- I1 in the mammary glands of mice, from the ovine P-lactoglobulin promoter, results in epithelial hyperplasia (Bates et al. 1995), whereas, overexpression of IGF- I1 from the moloney murine leukemia virus promoter results in delayed involution and apoptosis of mammary epithelial cell (MEC) and the sustained expression of Akt (Moorehead et al. 2001). The insulin-like growth factor- I1 receptor (IGF- I1 R) regulates the level or activity of numerous proteins, including the factors that control growth and differentiation. IGF- I1 R is more specific for IGF- I1 than for IGF- I and does not bind insulin. Although IGF- I1 R (the mannose-6-phosphate receptor) represents an exclusive receptor for IGF- 11, it targets IGF- I1 for lysosomal degradation without evoking a cellular response (Wise and Pravtcheva 2006). In addition, IGF- I R has generally been considered as the main pathway for IGF- I1 action, because IGF- I1 displays a low affinity for insulin receptor (IR) (Nielsen 1992). Indeed, IGF- I R is expressed in the normal mouse mammary gland during development (Richert and Wood 1999). However, several disparate results arising from mouse genetic models for IR, IGF- I R, and IGF- I1 have continued to imply a role for the IR in the IGF- I1 action. Recenty, some researches have displayed that IGF- I1 functions as an autocrine/ paracrine effector of prolactin (PRL) action in the normal mouse mammary gland. They have found that PRL induces transcription of IGF- I1 in MEC via a prolactin receptor (PRLR)-dependent pathway that leads to the stimulation of alveolar development. They have also found that PRL induces IRS-1 and IRS-2 in the mouse mammary gland, indicating a role for IGF- I1 as a local effector of PRL action during mammary gland development (Hovey et al. 2003). Published data point toward the role of the IGF
system during the developmental or apoptotic processes in the mammary gland. However, very little is known about their expression of protein level in the different stages. The goal of this descriptive study is to examine the expression pattern of IGF- I and - I1 and IGF- I R in the defined stages of mammary gland growth and lactation in mice. All the data should help in more experimentally oriented future studies, to discover the possible role of the IGF system in the mammary gland.
MATERIALS AND METHODS Animals, tissue samplings, and preparation For the pregnancy ages, adult Kunming female virgin mice were placed in mating and checked daily for vaginal plugs. Additional pregnant mice were allowed to deliver their pups for analyses of glands in lactation and involution. The morning when the vaginal plug appeared was considered as day 0. For lactation and involution studies, litters were culled or pups were cross-fostered on day 1, to normalize to eight pups per dam. In all studies, the number four abdominal glands were removed within 20 min after slaughtered in defined stages, and small pieces (1-2 g) of mammary tissue were flash frozen in liquid nitrogen for cryostat sectioning, and for immunofluorescence studies. At least three glands from individual mouse were analyzed for protein expression. Frozen cryostat sections (8-10 pm) were mounted onto slides coated with APES and stored at 4°C until use for immunofluorescence. The classification of the animals was established as follows: (1) virgin (D20, D30, D40, D50, D60, n = 3), (2) pregnancy (PO, P6, P12, P18, n=3), (3) lactation (Ll, L4, L8, L12, L16, L20, L24, L28, L32, n=3), (4) forced involution (remove litters from lactating mice at five-day lactation, L6&11, L7&12, L8&13, L9&14, n = 3).
lmmunofluorescence The presence of IGFs and IGF- I R was demonstrated by using the indirect immunofluorescence technique. Nonspecific protein binding was eliminated by incubation with 10% normal goat serum in PBS for 1 h at room
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The Expression of the IGF Family During Mouse Mammary Gland Development
temperature. Sections were then incubated overnight at 4°C with a goat polyclonal antibody to IGF- I (dilution 1:lOO) or a rabbit polyclonal antibody to IGF- 11 and IGF- I R (dilution 1:100), and for 1 h with FITCconjugated rabbit anti-goadgoat anti-rabbit IgG (diluted 1:200 in PBS-1% BSA). Between each step, sections were washed thrice in PBS. All incubations were carried out in humidified chambers to prevent evaporation. Sections were lightly counterstained in PI for 5 min at room temperature, diluted to I pg mL' in PBS. Controls were performed by replacing the primary antibody with normal IgG at the same or greater concentration.
Statistical analysis When the data subjected to one-way ANOVA showed significant differences, a Bonferroni test was performed.
RESULTS Mammogenesis (mammogenesis in virgin mouse) Mammogenesis in nonpregnant mice was mainly characterized by ductal growth. The mammary gland was characterized by a well-developed adipose tissue in which small groups of ducts could be seen. The condensed connective tissue surrounded the ductular epithelium. Between the ducts, areas of loose connective tissue were seen, which contained comparatively few cells. As shown in Fig. 1-A the interlobular stroma and the epithelium, where mammary cells aggregated, appeared positive for immunoreactive IGF- I . Many of the adipocytes also showed a positive IGF- I staining in their cytoplasm. As shown in Fig. 1-E, only the basal layer of the ductal epithelium was with strong immunostaining for IGF- I , whereas, the luminal layer of the ductal epithelium, the interlobular stroma, and adipocytes were negative or displayed only weak immunostaining for IGF- 11. As shown in Fig. 1-I, all compartments were positive for immunoreactive IGF- I R, but it was obviously stronger in the adipocytes than in the epithelium surrounding those cells along their cell membrane.
Alveolargenesis (days 1-18 of pregnancy of primigravid mouse) At the beginning of this period, the alveoli were well developed to lobuli. The alveoli were small, roundish and lined by a cuboidal epithelium, which appeared negative for immunoreactive IGF- I . The adipose tissue was well developed at the beginning and displayed a positive IGF- I staining. Later in this period, the number of adipocytes was reduced. The glandular epithelium was always negative for IGF- I . In the stroma, no significant changes were seen. Adipocytes were generally IGF- I positive in their cytoplasm. In late pregnancy, the amount of adipose tissue around the lobuli was further reduced, but the remaining adipocytes still appeared immunoreactive for IGF- I . No immunostaining for IGF- I was seen in the secretory epithelial cells. The alveolar epithelial cells appeared with a distinct positive staining for IGF- 11, and as shown in Fig.1-F, only the basal layer epithelium of the alveoli displayed IGF- I1 immunoreactive in early pregnancy. The alveoli in late pregnancy were still strongly positive for IGF- 11, but it was difficult to determine whether the positive staining was only in the basal layer epithelium. As shown in Fig.1-J, all compartments remained positive for immunoreactive IGF- I R with striking enhancement, especially in the epithelium of the acini.
Lactation (days 1-20of post-partum) The lobuli of the mammary gland consisted of numerous alveoli with wide lumina, separated by small amounts of connective tissue. Within the alveoli, a large amount of secretion occurred. As shown in Fig.1-C, the secretory epithelial cells showed a negative or weak staining for immunoreactive IGF- I . The interlobular stroma displayed weak immunonegative staining for IGF- I . The alveolar epithelial cells appeared with a distinct positive staining for IGF- 11 as shown in late pregnancy, with a distinctly strong staining on day four of lactation (Fig. 1-G). The positive staining for IGFI R was more stnking in lactation than any other period all round the whole slice, with an extremely strong staining on day 16 of lactation.
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Natural involution (days 21-32 of post-partum) After the mouse had lactated for 20 days, the appearance of the mammary parenchyma changed significantly. The alveoli were irregular in their outline and were lined by a secretory epithelium. Lobuli with small and roundish acini were distributed between areas, with large acini having irregular outlines and a folded epithelium. As in the previous stages, little IGF- I immunoreactivity was seen in the secretory cells of any of the acini or ducts. The number of weak IGFI -positive fat cells in the stroma was low. The density of the intralobular stroma had increased considerably and it stained weakly for IGF- I (Fig.1-D). A weak to distinct IGF- I1 immunoreactivity occurred in the alveoli, and in particular those depauperated alveoli were easily discerned by their pronounced staining for IGF11 (Fig. 1-H). IGF- I R positive staining remained distinct in the epithelium and the stroma, but predominantly in the adipocytes that reoccurred (Fig. 1-L).
Forced involution (24-96 h after forced weaning) 0-96 hours after weaning, since day 5 of lactation, the appearance of the mammary structure changed significantly as seen in the natural involuting process, but by a flash pattern. Ninety-six hours after weaning, the original alveolar structural of the mammary gland had completely regressed. The mammary gland completed a recombination on the whole. As in the previous stages, weak IGF- I immunoreactivity was seen in the mammary gland. Only in the fat tissue and the stroma, a few cells were weakly IGF- I -positive (Fig.2-A-D). A distinct to weak to distinct IGF- I1 immunoreactivity occurred in the alveoli, and in particular those depauperated alveoli were easily discerned by their pronounced staining for IGF- I1 (Fig. 2-E-H). IGF- I R positive staining remained distinct in the epithelium and the stroma, and it also had a distinct to weak to distinct pattern. 0-96 hours after weaning, IGF- I R was localized predominantly in the epithelium and the stroma. At 72 hours, IGF- I R-positive staining was obviously weak on the whole slice. At 96 hours, IGF- I R-positive staining was distinct again and predominant in the reoccurred adipocytes (Fig.2-I-L).
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Quantified analysis of protein levels of IGF family members’ expression in different development stages The patterns of IGFs and IGF- I R expression were examined by semi-quantified densitometric analysis of five pieces of corresponding laser scanning confocal microscope (LSCM) pictures, one of each of the three parallel groups. IGF- I slightly expressed at most stages, but was relatively stronger in the virgin. The highest IGF- I level was found in the mammary tissue of the day 50 virgin. The expression decreased significantly during lactation, followed by a slight increase in late lactation and involution (Fig.3-A and -D). The relative IGF- I expression of the virgin mice was obviously lower when compared to the IGF- I1 analysis. As shown in Fig.3-B and -D, IGF- I1 was strongly expressed in most stages, especially during middle pregnancy and early lactation. The highest IGFI1 level was found in the mammary tissue of day 4 lactation. Relative expression of IGF- I1 showed very significant differences in mammogenesis, pregnancy, lactation, and involution, and decreased significantly in early involution ( P <0.05). As shown in Fig.3-C and -E, the higher level of IGFI R expression was detected in late lactation (a clear increase appeared on day 16 of lactation) and the lower level in mammogenesis and early lactation. On the whole, the relative IGF- I R expression was obviously higher when compared with the other two analyses.
DISCUSSlON In general, the low abundance of IGF- I in almost all stages seems to suggest a minor function of the local IGF- I in the mouse mammary gland development, than endocrine IGF- I . It may be involved in duct morphogenesis and duct branching by a paracrine/ autocrine pattern in virgin mouse mammary gland. Local IGF- I , produced by mammary gland expresses both in the parenchyma and the stroma, showing its multifunctions. Epithelial-specific loss of IGF- I , during pubertal growth, results in deficits in ductal branching. In contrast, heterozygous reduction of IGF- I throughout the gland decreases expression of cyclins A2 and B1 during pubertal growth, and results in
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Fig. 1 Immunofluorescence analysis of IGF- I ,IGF- 11, and IGF- I R during normal mouse mammary gland development. PI counterstain. Magnification, 400 x. A-D, IGF- I in mouse mammary gland, D50, P6, L16, L28; E-H, IGF- TI in mouse mammary gland, D50, P6, L4, L28; I-L, IGF- I R in mouse mammary gland, D40,P6, L16, L28. Scale bars are about 50 pm.
Fig. 2 Immunofluorescence analysis of of IGF- I , IGF- 11, and IGF- I R in forced involuting mouse mammary gland. PI counterstain. Magnification, 400 x. A-D, IGF- I in mouse mammary gland, L6&11, L7&12, L8&13, L9&14; E-H, IGF- I1 in mouse mammary gland, L6&11, L78rI2, L8&13, L9&14; I-L, IGF- I R in mouse mammary gland, L6&11, L7&12, L8&13, and L9&14. Scale bars are about 50 pm.
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Fig. 3 Densitometric analysis of IGFs and IGF- I R in mouse mammary gland. Data from densitometric analysis are shown as means f SD (n=3). Significant differences (P<0.05) are indicated for IGF- I ,IGF- 11. and IGF- I R.
alterations in proliferation of the alveolar epithelium and milk protein levels during pregnancy-induced differentiation. Reduction in epithelial IGF- I at either of these stages has no effect on these indices (Richards et al. 2004; Loladze et al. 2006). Interestingly, a higher expression where mammary cells aggregate to form ducts indicates its actions involved in ductal morphogenesis in virgin mouse (Ruan and Kleinberg 1999; Ruan et al. 2005). The expression of IGF- I in adipocytes implies a potential effect of IGF- I on the proliferation and differentiation of adipocytes in this stage. The low level of IGF- I in pregnancy and lactation suggests that it is endocrine IGF- I which
regulates the mammary development in pregnancy and maintains milk production in lactation, and not local IGF- I . Both IGF- I and IGF- I1 have some similar functions for regulating mammary development alternatively or corporately. Similar to IGF- I , IGFI1 can also stimulate cell proliferation and be a survival factor of mammary epithelial cells in vitro, but with less potency than IGF- I (Pacher et al. 2007). IGFI1 mRNA has been detected in the mammary epithelial cells of both ruminants and rodents. The data further display that IGF- I1 level is obviously higher than IGFI in each mammary development stage, and increases
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The Expression of the IGF Family During Mouse Mammary Gland Development
dramatically in pregnancy and early lactation when compared to the constant low level in involution. These results indicate that locally produced IGF- I1 may play a greater role than locally produced IGF- I , with a potential positive action on alveolar morphogenesis and initiation of lactation. Hovey et al. (2003) had demonstrated that PRL enhances IGF- I1 mRNA expression, and IGF- I1 acted as the mediator of PRL in the mouse mammary gland during duct branching and alveolar development. From those data it is found that IGF- I1 may also act as a mediator of PRL on the onset of lactation, when PRL expression level is greatly upregulated, to initiate lactation (Tucker 2000; Freeman et al. 2000; Iwasaka et al. 2000). IGF- I1 is specially expressed in the basal layer epithelial cells of ducts and alveoli. It suggests that by a certain receptor or in an unknown manner, IGF- I1 is arrested in the basal layer epithelial cells membrane. It is impossible to answer for IGF- I R, because of positive staining for IGF- I R surrounding the cells along the membrane. It implies there is an IGF- I R-independent pathway for IGFI1 as is proved in pregnancy. As a survival factor of mammary epithelial cells, the decrease of IGF- I1 expression in involution following the weaning (natural or forced) may be essential to allow mammary cells apoptosis. The expression of IGF- I R is distinct in all stages examined, especially in early pregnancy (day 6 of pregnancy), and later lactation (day 16 of lactation). These results display that IGF- I R is still a vital receptor of IGFs during mammary gland development, even though there is a low abundance of local IGF- I and possible IGF- I R and independent action of IGF11. Some studies of transgenic mice that overexpress IGF- I during pregnancy and lactation h a v e demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation, but has minimal effect in early lactation on milk composition or milk production (Hadsell et al. 2002). These results show a distinct increase of IGF- I R in late lactation compared with those in the previously examined stages. It leads to the conclusion that IGF- I R-dependent pathways are involved in the maintenance of milk production in late lactation. This may be a direct effect of IGFs or of synergistic actions with other growth factors. In
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contrast, the low level of local IGF- I and the high expression of IGF- I R may suggest an increased importance of the endocrine IGF- I (produced by the liver), and may explain the well-known galactopoietic effect of exogenous GH. On the other hand, overactivation of IGF- I R in late lactation and within 48 h after weaning may result in loss of apico-basal polarity in mammary epithelial cells for involution as early lesions of breast cancer (Yanochko and Eckhart 2006). IGFI R-positive staining is distinctly localized in adipocytes and stromal cells, as well as the epithelial cells of ducts and acini. It indicates the significance of the IGF- I R-dependent pathway for the regulation of fat tissue and the stroma, in addition to the epithelium, during mammary gland development. In conclusion, the locally produced IGF- I and IGFI1 play a key role during mouse mammary gland development. IGF- I acts on the gland by IGF- I R. With a potential major function, in contrast to the former, the latter may act as a mediator of PRL action by an IGF- I R-independent pathway during lactation, which is the same as in mammogenesis and pregnancy. Given the high level of IGF- I R and the low abundance of local IGF- I in lactation, the action of endocrine IGF- I should be valued (Plath-Gabler et al. 2001). The increase of IGF- I R and the decrease of IGF- I1 following weaning can be important for the initiation of involution.
Acknowledgements This study was supported by Natural Science Foundation of Heilongjiang Province, China (ZTN0504).
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structure, function, and regulation of secretion. Physiological Reviews, 80, 1523-1631. Glait C, Tencer L, Ravid D, Sarfstein R, Liscovitch M, Werner H. 2006. Caveolin-1 up-regulates IGF- I receptor gene transcription in breast cancer cells via S p l - and p53dependent pathways. Experimental Cell Research, 312,38993908. Hadsell D L, Bonnette S G, Lee A V. 2002. Genetic manipulation of the IGF- I axis to regulate mammary gland development and function. Journal of Dairy Science, 85,365-377. Hadsell D L. 2003. The insulin-like growth factor system in normal mammary gland function. Breast Disease, 17,3-14. Hovey R C, Harris J, Hadsell D L, Lee A V, Ormandy C J, Vonderhaar B K. 2003. Local insulin-like growth factor- 11 mediates prolactin-induced mammary gland development. Molecular Endocrinology, 17,460-471. Ilkbahar Y N, Thordarson G, Camarillo I G, Talamantes F. 1999. Differential expression of the growth hormone receptor and growth hormone-binding protein in epithelia and stroma of the mouse mammary gland at various physiological stages. Journal of Endocrinology, 161,77-87. Iwasaka T, Umemura S, Kakimoto K, Koizumi H, Osamura Y R. 2000. Expression of prolactin mRNA in rat mammary gland during pregnancy and lactation. Journal of Histochemistry and Cytochemistry, 48,389-395. Loladze A V, Stull M A, Rowzee A M, DeMarco J, Lantry 111 J H, Rosen C J, LeRoith D, Wagner K-U, Hennighausen L, Wood T L. 2006. Epithelial-specific and stage-specific functions of insulin-like growth factor- I during postnatal mammary development. Endocrinology, 147,5412-5423. Moorehead R A, Fata J E, Johnson M B, Khokha R. 2001. Inhibition of mammary epithelial apoptosis and sustained phosphorylation of Akt/PKB in MMTV-IGF- 11 transgenic mice. Cell Death and DijJerentiation, 8, 16-29. Nielsen F C . 1992. The molecular and cellular biology of insulinlike growth factor 11. Progress in Growth Factor Research, 4, 257-290.
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