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SD Berry, RD Howard, PM Jobst, H Jiang and RM Akers

The objective was to determine the effects of ovariectomy and epithelial-stromal interactions on mammary development and local expression of IGF-I and IGF-binding protein (IGFBP) mRNA in prepubertal heifers. An epithelium-free ('cleared') fat pad (CFP) was prepared in two glands in each of 14 Holstein heifers, aged 1-3 Months. Eight of the calves were also ovariectomized. Serum concentrations of GH, IGF-I and prolactin were not affected by ovariectomy. At 6 Months of age, calves were killed to provide mammary samples of parenchyma, CFP and intact fat pad (MFP). Total mammary mass was reduced in ovariectomized calves (130+/-21 g vs 304+/- 25 g; P<0.001), and in several cases parenchymal tIssue was essentially absent. Uterus weight was also reduced by ovariectomy (14.5+/-3.8 g vs 30.4+/-4.5 g; P<0.05). In support of our hypothesis that local IGF-I mediates prepubertal mammary development, mRNA expression of IGF-I was lower in ovariectomized than in control calves (62.1+/-7.8 vs 91.6+/-7.8 arbitrary units; P<0.05). Specific binding of IGF-I to mammary parenchymal microsomes was also reduced by ovariectomy (377+/-142 vs 868+/-82 c.p.m.; P<0.01), suggesting decreased sensitivity to IGF-I. Expression of IGFBP-3 and IGFBP-5 mRNA were not influenced by ovariectomy. Expression of IGF-I, IGFBP-3 and IGFBP-5 mRNA did not differ between CFP and MFP, suggesting that expression of these factors was not influenced by interactions between stroma and developing epithelium. Overall, the data suggested that interactions between the ovary and the local IGF-I axis act to optimize the availability and effectiveness of IGF-I within the gland to stimulate mammary growth.

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MS Weber, S Purup, M Vestergaard, SE Ellis, J Scndergard-Andersen, RM Akers and K Sejrsen

Peripubertal development of the mammary gland is probably mediated by locally produced growth factors acting in concert with circulating mitogens. Our objective was to investigate the effect of recombinant human insulin-like growth factor-binding protein-3 (rhIGFBP-3) or insulin-like growth factor-I (IGF-I) antibodies on the IGF-I-related mitogenic activity of bovine serum and of mammary tissue extracts in primary mammary epithelial cell cultures. Cells were obtained from prepubertal female calf mammary tissue and cultured in three-dimensional collagen gels. An aqueous mammary parenchymal tissue extract (pooled from 20 prepubertal heifers) or serum (pooled from 3 heifers) at a concentration of 5% was added to the medium containing either rhIGFBP-3 or monoclonal or polyclonal antibodies to human IGF-I. Cell proliferation was evaluated using [methyl-3H]thymidine incorporation as a measure of DNA synthesis. Addition of mammary extracts stimulated DNA synthesis 545% compared with basal medium. Addition of serum stimulated DNA synthesis by 28%. Mitogenic activity of serum and added IGF-I was abolished by addition of rhIGFBP-3 in equimolar concentrations with IGF-I. For mammary extracts, 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 DNA synthesis by 26%, whereas IGF-I antibodies had no consistent effect. These results indicate that circulating and mammary-synthesized IGF-I and IGFBPs probably play a critical role in prepubertal development of the bovine mammary gland.

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GM Barrington, TE Besser, CC Gay, WC Davis, JJ Reeves, TB McFadden and RM Akers

Induction of colostrogenesis in non-pregnant cows was used to evaluate the relationship between prolactin (PRL) and mammary immunoglobulin G1 (IgG1) receptor expression. Six of eleven non-pregnant, non-lactating Holstein cattle responded to a standard lactation induction protocol by development of elevated IgG1 concentrations in mammary secretions. In order to increase the diversity in PRL concentrations, two of the six cattle were treated with bromocriptine, and two others were treated with recombinant bovine PRL. Serum alpha-lactalbumin, serum PRL and mammary secretion IgG1 concentrations were measured throughout the experiment. Biopsies of mammary tissue were collected after induction of lactation, and after treatments to alter serum PRL. Immunohistochemistry was used to evaluate IgG1 receptor expression. Administration of recombinant bovine (rbPRL) was associated with increased lactogenic activity, decreased secretion IgG1 concentrations, and decreased IgG1 receptor expression. Decreased serum PRL, due to bromocriptine, was associated with decreased lactogenic activity and maintenance of IgG1 receptor expression. Results of this experiment are consistent with an effect of PRL in decreasing the expression of the bovine mammary IgG1 receptor at the onset of lactogenesis.