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Nutritional and somatotropin regulation of the mitogenic response of mammary cells to mammary tissue extracts
Domestic Animal Endocrinology 18 (2000) 159 –164
Nutritional and somatotropin regulation of the mitogenic response of mammary cells to mammary tissue extracts M.S. Weberb, S. Purupa, M. Vestergaarda, R.M. Akersb,*, K. Sejrsena a
Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Foulum, DK-8830 Tjele, Denmark b Department of Dairy Science, Virginia Polytechnic Institute and State University, 2080 Litton Reaves Hall, Blacksburg, VA 24061-0315, USA Received 19 April 1999; accepted 30 August 1999
Abstract Our objective was to investigate the mitogenic response of primary mammary epithelial cells to extracts of mammary parenchyma from 24 prepubertal Friesian heifers treated with placebo or growth hormone at either a low or a high feeding level. The mitogenic responses to mammary extracts were tested by using primary mammary epithelial organoids obtained from prepubertal heifers cultured for 4 to 5 d in collagen gels in serum-free medium supplemented to 5% concentration of the mammary extracts. Cell proliferation was determined using [methyl-3H]thymidine incorporation as a measure of DNA synthesis. High feeding level reduced DNA synthesis in response to mammary extracts. At low feeding level, growth hormone treatment decreased DNA synthesis in response to mammary extracts whereas, at high feeding level, growth hormone increased DNA synthesis in response to mammary extracts. These results suggest that locally produced growth factors are involved in the regulation of mammary development when mammary growth is modulated by feeding level and growth hormone treatment. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Recent studies have increased our understanding of the roles of systemic and locally produced growth factors in mammary growth [1]. Production of at least some of these growth * Corresponding author. Tel.: ⫹1-504-231-4757; fax: ⫹1-504-231-5014. E-mail address: [email protected] (R.M. Akers) 0739-7240/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 9 9 ) 0 0 0 7 1 - 5
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factors such as the IGF is influenced by the stage of mammary gland development [2]. Further, levels of circulating growth hormone (GH) and local growth factors likely mediate at least a portion of the effects of both feeding level and exogenous GH on the mammary gland [3]. Presently, regulation of growth factor gene expression in the prepubertal bovine mammary gland is poorly understood. Moreover, complex interactions between hormones and metabolic regulators make it difficult to predict the growth response of the mammary gland by using in vivo experiments. The focus of this study was to determine whether the mitogenic activity in extracts of mammary tissue from prepubertal heifers was influenced by exogenous GH or feeding level differences.
2. Materials and methods 2.1. Experimental design Twenty-four Friesian heifers (233 ⫾ 7 kg b.wt., 271 ⫾ 4 d at slaughter) were randomly assigned within blocks of 4 to bST treatment at low or high feeding level in an experiment with a 2 ⫻ 2 factorial arrangement of treatments [4]. Treatments continued for 5 weeks and consisted of daily bST injections (0 or 100 mg/kg b.wt.) at either low or high feeding level. The low feeding level utilized a roughage-based mixed ration available ad lib and supported an average daily gain (ADG) of 0.55 kg/d. The high feeding level diet was a concentrate mixture available ad lib along with 2 kg/d of the mixed ration and supported an ADG of 1.1 kg/d. Mammary tissue was obtained at slaughter. Aqueous mammary extracts were prepared from thawed parenchymal tissue according to Weber et al. [5]. Tissue slices were homogenized for 1 min in physiological saline by using a Polytron tissue homogenizer (Kinematica, Buch, & Holm, Herlev, Denmark). The homogenate (3 : 1 saline to tissue) was shaken at 4°C for 90 min. Homogenates were filtered through two layers of gauze, and the filtrate was centrifuged for 45 min at 10, 000 ⫻ g at 4°C. The supernatant was recovered from below the uppermost fat layer and subsequently centrifuged at 105, 000 ⫻ g for 1 h at 4°C. The supernatant was filtered through sterile 0.2 m filters and stored at ⫺80°C. Protein content of the mammary extracts was determined using the bicinchoninic acid assay (Pierce Life Technologies, Roskilde, Denmark) and ranged from 8.24 to 15.35 mg/ml for the 24 extracts. Concentrations of IGF-I were measured by ELISA (OCTEIA IGF-I; IDS Ltd., Tyne and Wear, UK) [6] and bovine insulin by a TRFIA (DELFIA, Wallac, Turku, Finland). 2.2. Primary cell cultures Mammary epithelial cells used for evaluating the mitogenic response to extracts were prepared from mammary parenchymal tissue from two prepubertal Friesian heifers (198 and 223 kg b.wt.) and cultured in collagen gels according to Weber et al. [5]. Mammary parenchyma was excised aseptically from the outer margin of parenchyma. Tissue pieces were digested in basal medium supplemented with collagenase (10 mg/g tissue), hyaluron-
M.S. Weber et al. / Domestic Animal Endocrinology 18 (2000) 159 –164
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idase (10 mg/g tissue), DNase (277 g/g of tissue) and insulin (67 g/g of tissue), at a ratio of 10 ml medium per gram of tissue, for 5 h at 37°C. Organoids were isolated by filtering the suspension through 200 m Nitex and centrifuging the filtrate for 10 min at 300 ⫻ g. Organoids were suspended in basal medium and allowed to sediment for 8 min. Excess medium was aspirated and replaced with fresh medium for a total of 10 washes. Following suspension in basal medium supplemented with 44% FBS and 6% dimethyl sulfoxide (DMSO), organoids were frozen overnight at ⫺80°C and transferred the next day to liquid N2. Results for [methyl-3H]thymidine incorporation (Amersham International, Birkerød, Denmark) were determined on a liquid scintillation counter. Because variation was observed in the mitogenic response to basal medium among the five different assays (mean 92,123 ⫾ 5,186 d.p.m.), results were adjusted to a similar scale by dividing values for [3H]thymidine incorporation per well by the average basal value for incorporation in each assay. Results from five independent cell culture assays testing the effects of extracts on two different cell preparations were then averaged for analysis. 2.3. Statistical analysis Statistical analyses were performed using the GLM procedure of SAS. Results were analyzed as a randomized complete block design using block (sire groups) and treatments (GH, feeding level, and their interaction) as main effects, with interactions of block and treatments as the error term. Values are presented as least square means ⫾ SEM of values obtained from triplicate samples.
3. Results Addition of mammary extracts (5%) to culture medium stimulated [3H]thymidine incorporation (mean 594,159 ⫾ 20,967 d.p.m.) to a greater extent than did 10% fetal bovine serum (FBS; mean 348,561 ⫾ 28,063 d.p.m.) or 100 ng/ml of IGF-I (mean 218,068 ⫾ 21,143 d.p.m.). An earlier study showed a dose-dependent response to a pool of mammary extracts over the range of 2 to 8%, with maximal stimulation between 6 and 8% extract concentration in media [5]. A concentration near the middle of this range (i.e., 5%) was used for individual mammary extracts in cell cultures. Insulin concentrations in individual mammary extracts were undetectable. Concentrations of IGF-I in mammary extracts were 27.5, 28.9, 31.2 and 45.5 ⫾ 2.0 ng/ml for low-fed placebo, low-fed GH-treated, high-fed placebo, and high-fed GH-treated heifers, respectively. Growth hormone at high feeding level increased IGF-I concentration in mammary extracts (P ⬍ 0.001). High compared with low feeding level inhibited [3H]thymidine incorporation in response to mammary extracts from placebo-treated heifers (Fig. 1, P ⬍ 0.01). At the low feeding level, GH treatment decreased [3H]thymidine incorporation whereas GH at high feeding level increased [3H]thymidine incorporation in response to mammary extracts (Fig. 1, P ⬍ 0.05). Extract protein concentrations were not affected by heifer treatment (P ⬎ 0.10).
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Fig. 1. [3H]thymidine incorporation (d.p.m.) into primary cultures of bovine mammary epithelial organoids in response to media containing 5% extracts of mammary tissue from prepubertal heifers. Heifers were treated with or without GH at either a low feeding level or a high feeding level. Results were normalized by dividing values for [3H]thymidine incorporation per well by the average value for basal [3H]thymidine incorporation in each experiment. Values are LS means ⫾ SEM for cultures performed in triplicate using two different cell preparations. Responses were reduced with high feeding level (P ⬍ 0.01). Differences between placebo- and GH-treated heifers were significant (P ⬍ 0.05) at each feeding level.
4. Discussion Prepubertal growth and development of the mammary gland is dependent on circulating peptide and steroid hormones. It is evident from studies on normal ruminant mammary tissue that these hormones work in concert with locally synthesized tissue mediators to affect growth of the mammary epithelium. In the case of IGF-I, the growth response is modulated by IGFBPs that may also exert IGF-independent effects on growth [7]. Administration of GH to prepubertal heifers stimulates mammary development [8], prompting studies to identify the mediator(s) responsible for its effects on the mammary gland. In this study, GH treatment at low feeding level decreased the mitogenic response to mammary extracts. In contrast, at high feeding level, GH stimulated the mitogenic response to mammary extracts. Growth hormone treatment at high feeding level increased tissue IGF-I levels along with the mitogenic response to extracts, implying that locally available IGF-I may play a role in GH-induced mammary growth in heifers on a standard rate of gain [8]. However, at low feeding level, GH did not change the IGF-I concentration in tissue extracts. Thus, changes in IGF-I levels in mammary tissue do not seem to explain the effects of GH at low feeding level on the mitogenic response. It is likely that multiple growth factors synthesized in mammary tissue contribute to the overall mitogenic response. In conditioned medium from primary bovine mammary epithelial cells, the mitogenic activity was attributed to the effects of several growth factors including proteins of 6 kDa and less, and a small fraction related to the fibroblast growth factors [9]. The negative effect of a rapid growth rate on mammary gland development in prepubertal
M.S. Weber et al. / Domestic Animal Endocrinology 18 (2000) 159 –164
163
heifers has been well established [3], although the underlying mechanisms have been elucidated only in part. This study demonstrates that a rapid rate of gain reduces the mitogenic response to extracts of mammary tissue from prepubertal heifers compared with heifers gaining at a slower rate, suggesting that local changes in growth factor concentrations are important for growth of the mammary epithelium during this period of development. Levels of IGF-I in mammary tissue did not differ according to feeding level, unlike the mitogenic response. However, our earlier report indicated that a high feeding level increased IGFBP-3 levels in mammary tissue extracts from prepubertal heifers. Interestingly, the IGFBP-3 level in these mammary tissue extracts was negatively correlated (r ⫽ ⫺0.51, P ⬍ 0.02) with the mitogenic response to those extracts, suggesting a role for IGFBP-3 in the negative effect of a rapid growth rate [10]. Moreover, we have observed that addition of IGFBP-3 inhibits the stimulatory effect of IGF-I on mitogenesis [5]. Interactions between IGFBP-3 and locally produced growth factors in growth modulation are also supported by evidence for TGF--induced stimulation of IGFBP-3 synthesis in breast cancer cells and the demonstration that IGFBP-3 antisense oligonucleotides suppressed the inhibitory effect of TGF- on cell proliferation [11]. The presence of TGF-1 in serum [10] and mammary extracts (Purup et al., unpublished) from prepubertal heifers has been demonstrated. In conclusion, this study provides further evidence for the endocrine control of mammary synthesis of positive and negative-acting factors. Feeding level and exogenous GH can modulate prepubertal mammary development, potentially through mediation by locally produced factors. Elucidation of the interactive effects of these local factors will increase our understanding of the role of imbalanced expression of growth regulatory proteins in mammary gland development.
Acknowledgments The authors gratefully acknowledge L.R. Norup for assistance in cell culture experiments and Dr. R.E. Pearson for statistical advice.
References [1] Forsyth IA. The mammary gland. Bailliere’s Clin Endocrinol Metab 1991;5:809 –32. [2] Hovey RC, Davey HW, Mackenzie DDS, McFadden TB. Ontogeny and epithelial-stromal interactions regulate IGF expression in the ovine mammary gland. Mol Cell Endocrinol 1998;136:139 – 44. [3] Sejrsen K, Purup S. Influence of prepubertal feeding level on milk yield potential of dairy heifers: a review. J Anim Sci 1997;75:828 –35. [4] Vestergaard M, Purup S, Sejrsen K. Influence of bovine growth hormone and feeding level on growth and endocrine status of prepubertal heifers. J Anim Sci 1995;73(suppl. 1):148. [5] Weber MS, Purup S, Vestergaard M, Sondergaard–Andersen J, Akers RM, Sejrsen K. Contribution of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) to mitogenic activity in bovine mammary extracts and serum. J Endocrinol 1999;161:365–73. [6] Norup, LR, Vestergaard M. Use of the Octeia IGF-I assay for bovine and porcine samples. In: Norup LR, editor. Endokrin Regulering af Yverudviklingen Hos Kvier. Aarhus University, Denmark. MSc. Thesis, 1996.
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[7] Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocrine Rev 1995;16:3–34. [8] Purup S, Sejrsen K, Foldager J, Akers RM. Effect of exogenous bovine growth hormone and ovariectomy on prepubertal mammary growth, serum hormones, and acute in vitro proliferative response of mammary explants from Holstein heifers. J Endocrinol 1993;139:19 –26. [9] Sandowski Y, Peri I, Gertler A. Partial purification and characterization of putative paracrine/autocrine bovine mammary epithelium growth factors. Livest Prod Sci 1993;35:35– 48. [10] Purup S, Vestergaard M, Weber MS, Plaut K, Akers RM, Sejrsen K. Local regulation of pubertal mammary growth in heifers. J Anim Sci 2000;78(suppl 2). [11] Oh Y, Mu¨ller H, Ng L, Rosenfeld RG. Transforming growth factor--induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein-3 action. J Biol Chem 1995;270:13589 –92.
Nutritional and somatotropin regulation of the mitogenic response of mammary cells to mammary tissue extracts M.S. Weberb, S. Purupa, M. Vestergaarda, R.M. Akersb,*, K. Sejrsena a
Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Foulum, DK-8830 Tjele, Denmark b Department of Dairy Science, Virginia Polytechnic Institute and State University, 2080 Litton Reaves Hall, Blacksburg, VA 24061-0315, USA Received 19 April 1999; accepted 30 August 1999
Abstract Our objective was to investigate the mitogenic response of primary mammary epithelial cells to extracts of mammary parenchyma from 24 prepubertal Friesian heifers treated with placebo or growth hormone at either a low or a high feeding level. The mitogenic responses to mammary extracts were tested by using primary mammary epithelial organoids obtained from prepubertal heifers cultured for 4 to 5 d in collagen gels in serum-free medium supplemented to 5% concentration of the mammary extracts. Cell proliferation was determined using [methyl-3H]thymidine incorporation as a measure of DNA synthesis. High feeding level reduced DNA synthesis in response to mammary extracts. At low feeding level, growth hormone treatment decreased DNA synthesis in response to mammary extracts whereas, at high feeding level, growth hormone increased DNA synthesis in response to mammary extracts. These results suggest that locally produced growth factors are involved in the regulation of mammary development when mammary growth is modulated by feeding level and growth hormone treatment. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Recent studies have increased our understanding of the roles of systemic and locally produced growth factors in mammary growth [1]. Production of at least some of these growth * Corresponding author. Tel.: ⫹1-504-231-4757; fax: ⫹1-504-231-5014. E-mail address: [email protected] (R.M. Akers) 0739-7240/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 9 9 ) 0 0 0 7 1 - 5
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M.S. Weber et al. / Domestic Animal Endocrinology 18 (2000) 159 –164
factors such as the IGF is influenced by the stage of mammary gland development [2]. Further, levels of circulating growth hormone (GH) and local growth factors likely mediate at least a portion of the effects of both feeding level and exogenous GH on the mammary gland [3]. Presently, regulation of growth factor gene expression in the prepubertal bovine mammary gland is poorly understood. Moreover, complex interactions between hormones and metabolic regulators make it difficult to predict the growth response of the mammary gland by using in vivo experiments. The focus of this study was to determine whether the mitogenic activity in extracts of mammary tissue from prepubertal heifers was influenced by exogenous GH or feeding level differences.
2. Materials and methods 2.1. Experimental design Twenty-four Friesian heifers (233 ⫾ 7 kg b.wt., 271 ⫾ 4 d at slaughter) were randomly assigned within blocks of 4 to bST treatment at low or high feeding level in an experiment with a 2 ⫻ 2 factorial arrangement of treatments [4]. Treatments continued for 5 weeks and consisted of daily bST injections (0 or 100 mg/kg b.wt.) at either low or high feeding level. The low feeding level utilized a roughage-based mixed ration available ad lib and supported an average daily gain (ADG) of 0.55 kg/d. The high feeding level diet was a concentrate mixture available ad lib along with 2 kg/d of the mixed ration and supported an ADG of 1.1 kg/d. Mammary tissue was obtained at slaughter. Aqueous mammary extracts were prepared from thawed parenchymal tissue according to Weber et al. [5]. Tissue slices were homogenized for 1 min in physiological saline by using a Polytron tissue homogenizer (Kinematica, Buch, & Holm, Herlev, Denmark). The homogenate (3 : 1 saline to tissue) was shaken at 4°C for 90 min. Homogenates were filtered through two layers of gauze, and the filtrate was centrifuged for 45 min at 10, 000 ⫻ g at 4°C. The supernatant was recovered from below the uppermost fat layer and subsequently centrifuged at 105, 000 ⫻ g for 1 h at 4°C. The supernatant was filtered through sterile 0.2 m filters and stored at ⫺80°C. Protein content of the mammary extracts was determined using the bicinchoninic acid assay (Pierce Life Technologies, Roskilde, Denmark) and ranged from 8.24 to 15.35 mg/ml for the 24 extracts. Concentrations of IGF-I were measured by ELISA (OCTEIA IGF-I; IDS Ltd., Tyne and Wear, UK) [6] and bovine insulin by a TRFIA (DELFIA, Wallac, Turku, Finland). 2.2. Primary cell cultures Mammary epithelial cells used for evaluating the mitogenic response to extracts were prepared from mammary parenchymal tissue from two prepubertal Friesian heifers (198 and 223 kg b.wt.) and cultured in collagen gels according to Weber et al. [5]. Mammary parenchyma was excised aseptically from the outer margin of parenchyma. Tissue pieces were digested in basal medium supplemented with collagenase (10 mg/g tissue), hyaluron-
M.S. Weber et al. / Domestic Animal Endocrinology 18 (2000) 159 –164
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idase (10 mg/g tissue), DNase (277 g/g of tissue) and insulin (67 g/g of tissue), at a ratio of 10 ml medium per gram of tissue, for 5 h at 37°C. Organoids were isolated by filtering the suspension through 200 m Nitex and centrifuging the filtrate for 10 min at 300 ⫻ g. Organoids were suspended in basal medium and allowed to sediment for 8 min. Excess medium was aspirated and replaced with fresh medium for a total of 10 washes. Following suspension in basal medium supplemented with 44% FBS and 6% dimethyl sulfoxide (DMSO), organoids were frozen overnight at ⫺80°C and transferred the next day to liquid N2. Results for [methyl-3H]thymidine incorporation (Amersham International, Birkerød, Denmark) were determined on a liquid scintillation counter. Because variation was observed in the mitogenic response to basal medium among the five different assays (mean 92,123 ⫾ 5,186 d.p.m.), results were adjusted to a similar scale by dividing values for [3H]thymidine incorporation per well by the average basal value for incorporation in each assay. Results from five independent cell culture assays testing the effects of extracts on two different cell preparations were then averaged for analysis. 2.3. Statistical analysis Statistical analyses were performed using the GLM procedure of SAS. Results were analyzed as a randomized complete block design using block (sire groups) and treatments (GH, feeding level, and their interaction) as main effects, with interactions of block and treatments as the error term. Values are presented as least square means ⫾ SEM of values obtained from triplicate samples.
3. Results Addition of mammary extracts (5%) to culture medium stimulated [3H]thymidine incorporation (mean 594,159 ⫾ 20,967 d.p.m.) to a greater extent than did 10% fetal bovine serum (FBS; mean 348,561 ⫾ 28,063 d.p.m.) or 100 ng/ml of IGF-I (mean 218,068 ⫾ 21,143 d.p.m.). An earlier study showed a dose-dependent response to a pool of mammary extracts over the range of 2 to 8%, with maximal stimulation between 6 and 8% extract concentration in media [5]. A concentration near the middle of this range (i.e., 5%) was used for individual mammary extracts in cell cultures. Insulin concentrations in individual mammary extracts were undetectable. Concentrations of IGF-I in mammary extracts were 27.5, 28.9, 31.2 and 45.5 ⫾ 2.0 ng/ml for low-fed placebo, low-fed GH-treated, high-fed placebo, and high-fed GH-treated heifers, respectively. Growth hormone at high feeding level increased IGF-I concentration in mammary extracts (P ⬍ 0.001). High compared with low feeding level inhibited [3H]thymidine incorporation in response to mammary extracts from placebo-treated heifers (Fig. 1, P ⬍ 0.01). At the low feeding level, GH treatment decreased [3H]thymidine incorporation whereas GH at high feeding level increased [3H]thymidine incorporation in response to mammary extracts (Fig. 1, P ⬍ 0.05). Extract protein concentrations were not affected by heifer treatment (P ⬎ 0.10).
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Fig. 1. [3H]thymidine incorporation (d.p.m.) into primary cultures of bovine mammary epithelial organoids in response to media containing 5% extracts of mammary tissue from prepubertal heifers. Heifers were treated with or without GH at either a low feeding level or a high feeding level. Results were normalized by dividing values for [3H]thymidine incorporation per well by the average value for basal [3H]thymidine incorporation in each experiment. Values are LS means ⫾ SEM for cultures performed in triplicate using two different cell preparations. Responses were reduced with high feeding level (P ⬍ 0.01). Differences between placebo- and GH-treated heifers were significant (P ⬍ 0.05) at each feeding level.
4. Discussion Prepubertal growth and development of the mammary gland is dependent on circulating peptide and steroid hormones. It is evident from studies on normal ruminant mammary tissue that these hormones work in concert with locally synthesized tissue mediators to affect growth of the mammary epithelium. In the case of IGF-I, the growth response is modulated by IGFBPs that may also exert IGF-independent effects on growth [7]. Administration of GH to prepubertal heifers stimulates mammary development [8], prompting studies to identify the mediator(s) responsible for its effects on the mammary gland. In this study, GH treatment at low feeding level decreased the mitogenic response to mammary extracts. In contrast, at high feeding level, GH stimulated the mitogenic response to mammary extracts. Growth hormone treatment at high feeding level increased tissue IGF-I levels along with the mitogenic response to extracts, implying that locally available IGF-I may play a role in GH-induced mammary growth in heifers on a standard rate of gain [8]. However, at low feeding level, GH did not change the IGF-I concentration in tissue extracts. Thus, changes in IGF-I levels in mammary tissue do not seem to explain the effects of GH at low feeding level on the mitogenic response. It is likely that multiple growth factors synthesized in mammary tissue contribute to the overall mitogenic response. In conditioned medium from primary bovine mammary epithelial cells, the mitogenic activity was attributed to the effects of several growth factors including proteins of 6 kDa and less, and a small fraction related to the fibroblast growth factors [9]. The negative effect of a rapid growth rate on mammary gland development in prepubertal
M.S. Weber et al. / Domestic Animal Endocrinology 18 (2000) 159 –164
163
heifers has been well established [3], although the underlying mechanisms have been elucidated only in part. This study demonstrates that a rapid rate of gain reduces the mitogenic response to extracts of mammary tissue from prepubertal heifers compared with heifers gaining at a slower rate, suggesting that local changes in growth factor concentrations are important for growth of the mammary epithelium during this period of development. Levels of IGF-I in mammary tissue did not differ according to feeding level, unlike the mitogenic response. However, our earlier report indicated that a high feeding level increased IGFBP-3 levels in mammary tissue extracts from prepubertal heifers. Interestingly, the IGFBP-3 level in these mammary tissue extracts was negatively correlated (r ⫽ ⫺0.51, P ⬍ 0.02) with the mitogenic response to those extracts, suggesting a role for IGFBP-3 in the negative effect of a rapid growth rate [10]. Moreover, we have observed that addition of IGFBP-3 inhibits the stimulatory effect of IGF-I on mitogenesis [5]. Interactions between IGFBP-3 and locally produced growth factors in growth modulation are also supported by evidence for TGF--induced stimulation of IGFBP-3 synthesis in breast cancer cells and the demonstration that IGFBP-3 antisense oligonucleotides suppressed the inhibitory effect of TGF- on cell proliferation [11]. The presence of TGF-1 in serum [10] and mammary extracts (Purup et al., unpublished) from prepubertal heifers has been demonstrated. In conclusion, this study provides further evidence for the endocrine control of mammary synthesis of positive and negative-acting factors. Feeding level and exogenous GH can modulate prepubertal mammary development, potentially through mediation by locally produced factors. Elucidation of the interactive effects of these local factors will increase our understanding of the role of imbalanced expression of growth regulatory proteins in mammary gland development.
Acknowledgments The authors gratefully acknowledge L.R. Norup for assistance in cell culture experiments and Dr. R.E. Pearson for statistical advice.
References [1] Forsyth IA. The mammary gland. Bailliere’s Clin Endocrinol Metab 1991;5:809 –32. [2] Hovey RC, Davey HW, Mackenzie DDS, McFadden TB. Ontogeny and epithelial-stromal interactions regulate IGF expression in the ovine mammary gland. Mol Cell Endocrinol 1998;136:139 – 44. [3] Sejrsen K, Purup S. Influence of prepubertal feeding level on milk yield potential of dairy heifers: a review. J Anim Sci 1997;75:828 –35. [4] Vestergaard M, Purup S, Sejrsen K. Influence of bovine growth hormone and feeding level on growth and endocrine status of prepubertal heifers. J Anim Sci 1995;73(suppl. 1):148. [5] Weber MS, Purup S, Vestergaard M, Sondergaard–Andersen J, Akers RM, Sejrsen K. Contribution of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) to mitogenic activity in bovine mammary extracts and serum. J Endocrinol 1999;161:365–73. [6] Norup, LR, Vestergaard M. Use of the Octeia IGF-I assay for bovine and porcine samples. In: Norup LR, editor. Endokrin Regulering af Yverudviklingen Hos Kvier. Aarhus University, Denmark. MSc. Thesis, 1996.
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[7] Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocrine Rev 1995;16:3–34. [8] Purup S, Sejrsen K, Foldager J, Akers RM. Effect of exogenous bovine growth hormone and ovariectomy on prepubertal mammary growth, serum hormones, and acute in vitro proliferative response of mammary explants from Holstein heifers. J Endocrinol 1993;139:19 –26. [9] Sandowski Y, Peri I, Gertler A. Partial purification and characterization of putative paracrine/autocrine bovine mammary epithelium growth factors. Livest Prod Sci 1993;35:35– 48. [10] Purup S, Vestergaard M, Weber MS, Plaut K, Akers RM, Sejrsen K. Local regulation of pubertal mammary growth in heifers. J Anim Sci 2000;78(suppl 2). [11] Oh Y, Mu¨ller H, Ng L, Rosenfeld RG. Transforming growth factor--induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein-3 action. J Biol Chem 1995;270:13589 –92.