Tucker H L M, Beaudry K L, Parsons C L M, Ellis S E, Akers R M
Department of Dairy Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Biological Sciences Directorate, National Science Foundation, 4201 Wilson Boulevard, Arlington, VA 22230.
J Dairy Sci. 2016 Dec;99(12):10093-10101. doi: 10.3168/jds.2016-11550. Epub 2016 Oct 13.
Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.
青春期前乳腺发育涉及导管的延长和分支以及基质组织重塑。这一过程与卵巢和垂体激素、生长因子及局部调节因子密切相关。越来越多的证据表明，肌上皮细胞除了在射乳反射中具有公认的重要性外，在导管发育中也发挥作用。在有报道称青春期前小母牛卵巢切除后，肌上皮细胞随乳腺生长减缓而发生变化之后，我们对用抗雌激素他莫昔芬处理的青春期前小母牛乳腺组织中的肌上皮细胞进行了评估。简要来说，小母牛从出生第28天开始接受安慰剂（n = 7）或他莫昔芬（n = 8；每天0.3mg/kg）处理，直至在第120天对动物实施安乐死。从3个区域（腺泡池附近、腺泡池与乳腺脂肪垫中间位置以及实质与乳腺脂肪垫交界处）分别采集组织。对样本进行处理以测量转化相关蛋白63（p63）、平滑肌肌动蛋白和普通急性淋巴细胞白血病抗原的表达。我们发现平滑肌肌动蛋白和普通急性淋巴细胞白血病抗原在肌上皮细胞的细胞质中表达，而p63在细胞核中表达。与他莫昔芬处理后乳腺生长受损50%相一致，我们发现发育中的乳腺导管周围的肌上皮细胞数量减少。但每个细胞核中p63表达的平均强度未受影响。我们利用p63在肌上皮细胞核中非常独特且专一的染色，采集了数百个细胞核图像，以便随后使用CellProfiler软件进行分析。通过该图像分析，我们发现他莫昔芬使肌上皮细胞核的面积和周长减小。当根据核形状（偏心率）对细胞核进行分类时，基于泽尼克数评估，我们发现面积、周长以及p63表达模式存在差异，并且在每种形状分类中也存在处理差异。这些数据支持了以下概念：肌上皮细胞也参与青春期前牛乳腺的发育，并且多光谱成像结合图像分析软件的应用能够提供定量数据，以更好地理解最终调节牛乳腺形态发生的复杂细胞相互作用。
