Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy.

The objectives of the experiment were (1) to determine whether MAC-T cells would accurately mimic the previously observed proliferative responses of primary mammary epithelial cells (MEC) to mammary tissue extracts from high and low-fed heifers and (2) to determine whether mammary tissue extracts from ovariectomized (OVX) heifers would have lower mitogenic activity than intact controls. Addition of mammary tissue extracts to cell culture media of MAC-T cells plated on plastic or collagen-coated plastic to a range of concentrations between 1 and 8% resulted in dose-dependent increases in cell proliferation. Furthermore, mammary tissue extracts from low-fed prepubertal heifers aged 9 months, stimulated significantly more proliferation of MAC-T cells, as measured by 3H-thymidine incorporation into DNA than mammary tissue extracts from high-fed heifers (40.6 cpm x 10(3) per well versus 21.9+/-1.8 cpm x 10(3) per well). These observations suggested that MAC-T cells would be a suitable alternative to primary MECs for measuring the mitogenic activity of mammary tissue extracts. Conversely, no difference was observed in the mitogenic activity of mammary tissue extracts from OVX or control heifers. Possibly, MAC-T cells provide a good model for nutrition- but not ovarian-induced changes in mammary growth. Alternatively, that reduction of in vivo mammary development following OVX did not result in reduced mitogenic activity of the mammary tissue extracts emphasizes that heifer mammary development is the result of complex interactions between local growth factors and systemic hormones.

Mammary localization and abundance of laminin, fibronectin, and collagen IV proteins in prepubertal heifers.

The objective was to determine localization and abundance of extracellular matrix proteins fibronectin, laminin, and collagen in mammary tissues from ovariectomized or intact prepubertal heifers. Mammary parenchyma and fat pad tissues were collected from 14 6-mo-old heifers: eight were ovariectomized between 1 to 3 mo of age, and six were used as intact controls. Distribution of total collagen was assessed by Sirius Red staining of tissue sections. Fibronectin, laminin, and type IV collagen were assessed by immunohistochemistry. Abundance of fibronectin and laminin was also analyzed by western blotting. Total mammary mass was much less in ovariectomized animals (130 +/- 21 vs. 304 +/- 25 g). Histological structure differed as parenchyma from intact animals contained abundant, complex branching epithelial terminal ductular units, whereas terminal ductular units from ovariectomized animals were mostly major ductal structures with little or no branching. Collagen fibers were abundant and densely packed throughout interlobular stroma and were less abundant and more diffuse within intralobular stroma. Type IV collagen was primarily in basal lamina of mature ducts, whereas fibronectin and laminin staining were present throughout parenchymal stroma, in both intact and ovariectomized animals. Using western blotting, fibronectin was more abundant within parenchyma than in the fat pad and significantly higher in parenchyma from ovariectomized heifers. Laminin was more abundant in parenchyma from intact than ovariectomized animals (30 vs. 17 densitometric units/mg of tissue), but laminin was similar between parenchyma and fat pad. These results provide initial evidence that fibronectin, laminin, and collagen participate in regulation of heifer prepubertal mammary development.

Mammary epithelial proliferation and estrogen receptor alpha expression in prepubertal heifers: effects of ovariectomy and growth hormone.

The objectives of this study were to determine the effects of ovariectomy and growth hormone on mammary epithelial cell proliferation and estrogen receptor alpha (ER alpha) expression within the bovine mammary gland. Two experiments were performed. In the first experiment, eight Holstein heifer calves aged between 1 and 3 mo were ovariectomized, while six calves served as controls. At 6 mo of age, calves were treated with bromodeoxyuridine (BrdU) to label proliferating cells and sacrificed 2 h later. Coinciding with reduced mammary mass (304 +/- 25 vs. 130 +/- 21 g), proliferation of mammary epithelial cells was significantly lower in ovariectomized heifers compared to control heifers (2.24 vs. 0.25%). ER alpha expression was restricted to mammary epithelial cells and was not observed within intra-lobular stroma of parenchymal tissue. The proportion of ER alpha positive cells was significantly higher in ovariectomized heifers than in controls (36.1% +/- 2.2 vs. 46.7% +/- 2.4). In the second experiment, mammary biopsies were taken from five 6-mo-old heifers, immediately preceding and 7 d following a single injection of bovine growth hormone. Mammary epithelial cell proliferation (assessed by incorporation of 3H-thymidine) was increased by growth hormone. The proportion of ER alpha positive mammary epithelial cells was not increased by growth hormone. In conclusion, reduced mammary epithelial cell proliferation following ovariectomy was associated with an increase in ER alpha expression, whereas increased proliferation caused by bovine growth hormone was not associated with changes in the proportion of ER alpha positive cells.

Interactions between the ovary and the local IGF-I axis modulate mammary development in prepubertal heifers.

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.