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Contribution of insulin-like growth factor (IGF)-I and IGF-bindingprotein-3 to mitogenic activity in bovine mammary extractsand serumM S Weber 1 , S Purup, M Vestergaard, S E Ellis 1 ,J S ¢ ndergård-Andersen 2 , R M Akers 1 and K SejrsenDepartment of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Foulum, DK-8830 Tjele, Denmark1 Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061–0315, USA2 Department of Analytical Chemistry, Novo Nordisk A/S, DK-3250, Gentofte, Denmark(Requests for offprints should be addressed to R M Akers)AbstractPeripubertal development of the mammary gland isprobably mediated by locally produced growth factorsacting in concert with circulating mitogens. Our objectivewas to investigate the effect of recombinant humaninsulin-like growth factor-binding protein-3 (rhIGFBP-3)or insulin-like growth factor-I (IGF-I) antibodies on theIGF-I-related mitogenic activity of bovine serum and ofmammary tissue extracts in primary mammary epithelialcell cultures. Cells were obtained from prepubertal femalecalf mammary tissue and cultured in three-dimensionalcollagen gels. An aqueous mammary parenchymal tissueextract (pooled from 20 prepubertal heifers) or serum(pooled from 3 heifers) at a concentration of 5% was addedto the medium containing either rhIGFBP-3 or mono-clonal or polyclonal antibodies to human IGF-I. Cellproliferation was evaluated using [methyl- 3 H]thymidineincorporation as a measure of DNA synthesis. Addition ofmammary extracts stimulated DNA synthesis 545% com-pared with basal medium. Addition of serum stimulatedDNA synthesis by 28%. Mitogenic activity of serum andadded IGF-I was abolished by addition of rhIGFBP-3in equimolar concentrations with IGF-I. For mammaryextracts, mitogenic activity was inhibited by 35%, 50%,and 82% by the addition of rhIGFBP-3 at, respectively, 1,2 and 4 times the molar IGF-I concentration in the extract.Addition of rhIGFBP-3 to basal medium reduced DNAsynthesis by 26%, whereas IGF-I antibodies had noconsistent effect. These results indicate that circulating andmammary-synthesized IGF-I and IGFBPs probably play acritical role in prepubertal development of the bovinemammary gland.Journal of Endocrinology (1999) 161, 365–373IntroductionRegulation of mammary growth and development isaccomplished by a complex interplay of circulatinghormones with locally acting growth factors. Classicstudies in laboratory and domestic species established theinvolvement of hormones from the ovary and pituitarygland in hormonal control of mammary development(Forsyth 1991). Subsequent investigations into the mech-anisms underlying the effects of these hormones onmammary cell growth revealed the existence of potentsystemic as well as mammary-derived growth factors thatmay mediate much of their activity (Oka et al. 1991).Moreover, Waksman et al. (1991) demonstrated that tissueextracts from bovine pituitary, kidney, uterus and mam-mary gland stimulated mammary cell DNA synthesis in adose-dependent manner and concluded that normal mam-mary epithelial cell proliferation is probably influenced bya multitude of tissue-derived growth factors.Exogenous bovine somatotropin (bST) stimulatesperipubertal mammary growth in dairy heifers (Sejrsenet al. 1986) and sheep (McFadden et al. 1990). However,much evidence indicates that bST functions by an indirectmechanism of action, mediated at least in part by insulin-like growth factor (IGF)-I (Akers 1985, Kleinberg 1997).The mitogenic effect of IGF-I on mammary explants(Baumrucker & Stemberger 1989, Purup et al. 1995), ormammary organoids of bovine (Shamay et al. 1988,McGrath et al. 1991, Purup et al. 1995) and ovine (Winderet al. 1989) origin in collagen gel culture, and in mammarytissue of pregnant heifers (Collier et al. 1993) has beendocumented. In the mammary gland, IGF-I is synthesizedlocally by stromal tissue and is considered to act onadjoining epithelial cells via a paracrine mechanism ofaction (Cullen et al. 1992). It is likely that IGF-I ofboth systemic and local origins influences mammary cellproliferation, together with additional serum- andmammary-derived growth factors.365Journal of Endocrinology (1999) 161, 365–3730022–0795/99/0161–365 ? 1999 Society for Endocrinology Printed in Great BritainOnline version via http://www.endocrinology.org