Cell suspensions containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone-responsive mammary tumors from animals treated with dimethylbenzanthracene. Cell suspensions were fractionated into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific celljunctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from nonneoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10% serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin was relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.Growth and development of the mammary gland in mice and rats occur mainly by a process of cell multiplication rather than by hypertrophy of existing cellular units (9). The hormones which control this process in vivo have largely been identified by means of a series of endocrine gland-ablation and hormone-replacement experiments, and they include prolactin, growth hormone together with estrogens, glucocorticoids, and progesterone (18,21,34,35). In organ culture, however, Topper and others have failed to show any mitogenic effect of prolactin or growth hormone although high, nonphysiological concentrations of insulin and a corticoid were shown to stimulate thymidine incorporation into DNA (19,22,33,38,40). Since pretreatment of virgin mice with prolactin
0 R o \ 147, Livcrpx)l, L69 3BX, UK. and 'Hannah Rescarch Institutc. Ayr. KA6 SHL, UK.In order to understand thc intercellular prtmsscs governing m a m n i q growth in the ruminant mammary gland we have examined the histology and immunophenotypic properties ot cclls in gtut mammary tissuc at different stages of development. This study cmploycd standard histochemical and immuntxyttxhemical techniques with immunological markers of ccll typcs which have previously been used in the study of rtdcnt I I I and human 121 mammary tissue.Mammary tissuc was obtained from goats at key stages 01-mammary development: virgin (6 months of age); pregnant (5 wccks pre-partum); lactaong (2nd wcck of twice daily milking); involuting (3rd week after ccssation of milking). Tissues were lixed in Mtdificd Mcthacam ((50% methanol, 30%) Inhibisol, 10% acetic acid; v h ) and prtxesscd and embedded in paraffin wax using standard histological prclcedurcs. Sections wcrc cut aid stained with haematoxylin and cosin or immunocyttxhemically s t a n d using the ABC mcthtxl (DAKO, High Wycombe, UK).The ductal stmcture in a &month cid virgin g t~t was traced and identified in scnal histological w t i o n s of mammary parenchyma. 'l'hc ductal system culminated in terminal ductallobular units (TDLU) which were comprised of lobules of blindending ducts or ductules (DTL). These DTLs wcrc connected to intmlobular terminal d u c~s that were draned by extralobular terminal ducts. The structure of the DTLs varied according to the physiological status of the gtat. In resting glands, the majority of DTLs wcrc in the form of alvetJar buds which expanded during gestation to form grape-like lobules of alveoli. In lactating glands, dilation of DTLs was at a maximum forming true sccrettw)? alvetii. All ducts and DTLs were lined by 1-3 layers of cuboidal, (or columnar in lactating glands), epithelial cells which. in turn, were surrounded by a single layer of myoepithelial cclls. The involuting gland was chardcterised by the wilapse of alvecdi causal by the loss of columnar mtwphtdogy of the alveolar cells which resulted in mytqnthelial cells being mtxc pmminent. Large numbers of body defence cells had infiltrated the parenchyma during involution.Immumxyttlchemical staining was performed to identify and investigate the histogenesis of epithelial, myoepthelial and al\8et>lar cells. The cxprcssion o f cytokeratins wa used a s an indicator of histogenetic differentiation of which cytokeratin 18 w m found in both cpithclial and mycxpthelial cells of the goat mammary gland. A decreasc in the number of cytcdceratinexpressing epithelial cells was observed in pregnant alveoli. No staining was o b s e n d in alveolar cells, although cytokeratin 18 w i~s lound in cclls, distinct from thc mycxpithclial phenotype, in alvetdi during lactation.This study demonstrates that the mammary gland of the goat is similar LO the pnmate, rather than rodent, mammary gland. This conclusion is justilicd by the idcntilication of similar ductal structures and TDLUs found in thc pnmate mammary gl...
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