Normal human breast tissue was enzymatically dissociated and the cells were injected into the gland-free fat-pads of athymic nude mice. Within 30 days, small, spherical, duct-like epithelial elements (organoids) formed in 68% of the fat-pads inoculated (0-23 organoids/fat-pad). Short-term (30-day) treatment of the host mice with mammotrophic hormones [secretions from a chorionic, soamto-mammotrophin-secreting transplantable human choriocarcinoma (JEG-3), secretions from a prolactin- and growth-hormone-secreting transplantable rat pituitary tumor (GH3), estrogen and/or progesterone] and/or cAMP inducers (cholera toxin) significantly (p less than 0.05) increased the size of the human breast organoids but did not increase organoid number or induce extensive and expansive growth (extensive duct elongation and branching) of these structures. Such treatments induced intense proliferation of the host mouse mammae resembling that which occurs during late pregnancy. The results of this study, therefore, provide evidence that normal human breast epithelium can be readily accepted by and maintained in the gland-free fat-pad of the athymic nude mouse, and the epithelium, within 30 days, forms spherical duct-like structures (organoids). The human breast organoids are hormone-responsive, as they respond to a mammotrophic growth stimulus by an increase in size. The failure of the human breast organoids to grow expansively in the gland-free fat-pad of this immunologically deficient mouse does not appear to be due to the absence of an appropriate hormonal growth stimulus.

Multiparous Holstein cows were infected in two quarters by intramammary infusion with Streptococcus agalactiae and slaughtered approximately 36 h later. Mammary tissue was removed from the infected quarters, uninfected contralateral quarters, and from pair-slaughtered uninfected controls; the tissue was frozen in liquid nitrogen. The RNA was extracted, and Northern blot analysis was performed for a variety of growth factors, stress-induced genes, milk protein genes, and control genes. Infection increased levels of mRNA coding for heat shock proteins 89 alpha, 89 beta, 70, 60, and 27. Simultaneously, concentrations of alpha-lactalbumin and casein mRNA decreased; alpha-lactalbumin mRNA showed a greater decline. The mRNA for several growth factors, including acidic fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, IGF-I, and IGF-II, were also increased as was the apoptosis marker, testosterone-repressed prostate mucin-2. Concentrations of mRNA for controls, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase were unaffected. These results indicate that mastitis induces changes in the levels of mRNA encoding for a variety of peptide growth factors. Such changes in growth factors could be important in a variety of processes that occur during infection, such as protection against injury or tissue repair and recovery processes.

Mammary gland development consists of a series of very highly ordered events involving interactions among a number of distinct cell types. An important aspect of mammary gland development is that the mammary gland consists of a fat pad of mesodermal origin into which epithelial cells of ectodermal origin proliferate. This proliferation of epithelial cells into the mammary fat pad is the subject of this review. The nature of the stroma into which epithelial cells proliferate is of considerable importance in determining the structure of the resulting gland. In mice, white adipose tissue appears to be required for normal mammary development. Transplantation of mammary epithelia to other types of stroma does not support epithelial growth or result in abnormal growth. To date, a synthetic substratum capable of mimicking white adipose tissue has not been developed. Although collagen gel cultures are generally considered superior to glass or plastic substratum in supporting near normal epithelial growth, the technique has not advanced to the point that the in vivo growth pattern is duplicated. Recent research on the generation of chimeric mammary tissue (by transplanting mammary epithelia from rats, cows, and women to the mammary fat pads of athymic nude mice) suggests that there are important species differences in the stromal requirements for mammary gland development. In particular, extensive and expansive growth of rat mammary tissue is observed in mouse mammary fat pads. However, the mouse mammary fat pad appears incapable of supporting expansive growth of bovine or human mammary epithelia. The reason for this difference is not clear. However, human and bovine mammary epithelia may require the presence of more fibrous (collagenous) tissue than rodent mammary epithelia for normal proliferation.

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Mammary epithelium from five Holstein cows was transplanted as subcutaneous slices and as collagenase dissociated epithelial cells into mammary gland free-fat pads of athymic nude mice. Two weeks posttransplantation, mice were injected daily for 20 d with saline, 17 beta-estradiol plus progesterone, or 17 beta-estradiol plus progesterone plus growth hormone plus prolactin. In a second experiment, mice were treated with saline, cholera toxin, 17 beta-estradiol (subcutaneous pellet of 2 mg 17 beta-estradiol and 38 mg cholesterol), or 17 beta-estradiol plus cholera toxin. In each experiment mammary slices maintained normal morphology. Growth of epithelium within slices ([3H]thymidine autoradiography) was increased 167% by estradiol plus progesterone, 264% by estradiol plus progesterone plus growth hormone plus prolactin, 90% by estradiol, 60% by cholera toxin, and 137% by estradiol plus cholera toxin. Cells injected into mammary gland free fat pads formed hollow, multilayered, spherical "organoids." Organoid area was increased 47% by estradiol plus progesterone, 189% by estradiol plus progesterone plus growth hormone plus prolactin, 72% by estradiol, 86% by cholera toxin and 74% by estradiol plus cholera toxin. Thus, athymic nude mice appear suitable for the ex vivo in vivo study of bovine mammary epithelial growth and differentiation.