Perfluorooctanoic acid (PFOA) is one of the most common man-made long-lasting environmental pollutants. This chemical is found in foods, drinking water, some cookware, and industrial products. It leads to tumors in the liver, pancreas, and testicles of mice and rats. Most human studies on the health effects of PFOA contact involve adults. Not long ago, high levels of PFOA were found in a group of 6 to 8 year old girls. However, it is not clear whether this will have harmful health effects in the future. It is generally believed that a young girl is more easily affected by pollutants before she starts her period than after that time. To study the possible health effects of PFOA that might be linked to breast cancer risk, we did PFOA contact studies in young female mice before their first period. Two kinds (strains) of mice that had different genetic make-ups are used. One is called the C57BL/6 strain and the other is the BALB/c strain. We found that contact with PFOA had opposite effects on the mammary glands (breasts) of the two strains. In the C57BL/6 strain, PFOA made the breast grow larger and faster than normal. In the BALB/c strain, the breasts grew more slowly or not at all. This finding might have to do with the impact of PFOA on the ovaries. PFOA contact seemed to stop the role of the ovaries in BALB/c mice. It did not show a harmful impact on the role of the ovaries for the C57BL/6 mice.

The different effects of PFOA between the two strains are a key finding. It shows that a person’s genetic make-up is likely central in the effects of PFOA.

The hormone progesterone (P) acting through its receptor, the progesterone receptor (PR), promotes development of the normal mammary gland and is also involved in the development of breast cancer. We used a cell culture system with organoids, clusters of mammary gland epithelial cells, grown on a three-dimensional matrix to identify genes whose expression was increased or decreased by P. The organoids were isolated from normal pubertal and adult mouse mammary glands. Puberty and adulthood are stages of mammary gland development with different P responsiveness. In this organoid system, P acts through progesterone receptor A (PRA), the major form of PR expressed in mammary organoids. We used a technique called microarray analysis to analyze known genes in the mouse for changes in expression in response to P. Our analysis showed significant progestin regulation, up or down, of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. We observed greater increases in the adult organoids in the expression of two mammary signaling factors, receptor activator of NFkappaB ligand and calcitonin, suggesting possible roles for these two factors in the different P responsiveness of the adult and pubertal mammary glands. We also examined the progestin-regulated genes for association with certain biological processes, and we found a significant increase by P in genes associated with cell adhesion, immune response, and survival. Striking increases in the expression of genes involved in innate immunity processes were noted in the organoid system and it was also confirmed that these genes were regulated similarly in vivo in the mouse mammary gland. These studies revealed new targets of PRA in mammary epithelial cells and a new link between P and inflammation during mammary gland development.

Progesterone (P) is an important hormone for normal development of the mammary gland and also influences the formation of breast cancer in rodent models and in humans. In this study, we analyzed mammary gland responses to P and estrogen (E) during normal development in two strains of mice with different genetic backgrounds (BALB/c and C57BL/6). The mammary gland in these two mouse strains develops differently and have different sensitivity to hormones. We found the mammary gland in the C57BL/6 mouse strain had a reduced response to P compared to the BALB/c mouse strain. We examined proteins known for having an effect on mammary gland development and that are controlled by P and found a number of proteins whose levels were reduced or whose location in the cell was altered in the mammary gland of the C57BL/6 strain. In contrast, the C57BL/6 mouse strain had a greater response to estrogen. These results suggest that in human populations where there is a lot of genetic variability, individuals may respond quite differently to the same hormone. Therefore, genetic background likely has an important role in determining the relative effects of estrogen or progesterone during normal mammary gland development and tumor formation.

Stat5a is a protein that is important for controlling growth and development of the mammary gland, and has also been implicated in breast cancer.  Stat5a functions as a transcription factor that induces expression of genes, but in order to do so it must first be activated. In mammary cells, Stat5a is activated primarily via the hormone prolactin.  Although the mechanisms that activate Stat5a are well understood, very little is known about those that regulate its expression in the mammary gland.  In this report, sections of mouse mammary glands were stained for Stat5a protein in order to examine its levels throughout development.  We found that it is not present before puberty, when estrogen (E) and progesterone (P) levels are low, but appears during puberty and is maintained in adult animals. When both E and P were eliminated from mice by removing the ovaries, Stat5a disappeared, and treatment with E+P was necessary to restore its expression to the level seen in intact, mature animals.  Stat5a positive cells also contained receptors for both E and P, suggesting that its expression is directly regulated by these receptors in response to hormone treatment.  Thus, these results identify a novel mechanism in which E and P induce Stat5a expression, allowing for the protein to be subsequently activated by prolactin. The active protein is then able to regulate downstream genes in order to promote mammary gland development.

Progesterone acting through two isoforms of the progesterone receptor (PR), PRA and PRB, regulates proliferation and differentiation in the normal mammary gland in mouse, rat and human. Progesterone and PR have also been implicated in the etiology and pathogenesis of human breast cancer. The focus of this review is on recent advances in understanding the role of the PR isoform specific functions in the normal breast and in breast cancer. Also discussed is information obtained from rodent studies on the hormonal regulation of PR isoform expression in mammary gland, recent progress in unraveling the molecular mechanisms of P action via PR isoforms, comparison of human, mouse, and rat PR isoform expression, and possible relationship of PR isoform expression to normal mammary gland development in human, rat, and mouse. Studies of P action and PR isoform-specific functions in normal mammary gland and during tumor development in animal models can provide an important insight into breast cancer etiology.  Most importantly, such studies may provide novel preventive, diagnostic, and therapeutic strategies to fight breast cancer.

In the normal mouse mammary gland progesterone (P) can cause cells to divide or change form through two progesterone receptor (PR) isoforms, PRA and PRB. PRA is the predominant isoform expressed in the adult virgin mouse, and PRB is predominantly expressed during pregnancy. In order to study the control of the PR isoforms by hormones and to examine the different functions of PRA and PRB, we removed the ovaries, which are the major source of hormone production, from adult mice and the mice were treated for 3, 5, or 10 days with estrogen (E), P, or estrogen + progesterone (E+P). We used a technique called immunohistochemistry to stain thin sections of mammary tissue with antibodies specific for PRA or PRB. This technique allowed us to investigate the regulation of PRA and PRB by hormones and to determine the role of PRA and PRB in cell turnover and overall changes in organization of the mammary gland. E treatment caused limited cell turnover that was only present after 5 days and was localized to the ends of ducts. P-induced cell turnover led to the formation of small buds, called sidebranches, off of the ducts and formation of grape-like clusters of cells called alveoli. However, the effect of E+P on sidebranching and formation of alveoli was greater. E increased PRA level, while P decreased PRA level. PRB expression was only detected following treatment with P or E+P. During sidebranching, PRA was the predominant PR isoform expressed. Cell turnover of both PRA expressing and PRA negative cells was responsible for P-induced sidebranching. In contrast, PRB was the predominant PR isoform expressed during formation of alveoli. Cell turnover of both PRB expressing and PRB negative cells was responsible for P-induced alveolar expansion. These results demonstrate that control of PRA and PRB levels by hormones in vivo occur differently and suggest that P induces cell turnover through PRA and PRB by direct and indirect mechanisms.

Progesterone (P), acting through progesterone receptor (PR) isoforms A and B, plays an important role in normal mammary gland development and is implicated in the etiology of breast cancer. Because of notable similarities between human and rat mammary gland development and hormonal responsiveness of mammary cancers in both the human and rat we undertook the analysis of progesterone action in the rat mammary gland. Using antibodies that detect only PRA or only PRB by immunohistochemistry, we investigated PRA and PRB expression at various mammary gland developmental stages (puberty, sexual maturity, pregnancy, lactation and after postlactational involution), and their functional roles in the regulation of proliferation. The percentage of PRA positive (PRA+) cells decreased from puberty to adulthood and further decreased after pregnancy, the percentage of PRB expressing cells was relatively constant at all developmental stages. Interestingly, during all developmental stages there was a significant proportion of cells that expressed only PRB. We found that the majority of PRA+ cells co-expressed PRB.  In the pubertal and adult virgin mammary gland, PRA+PRB+ cells also expressed nuclear cyclin D1, protein that is essential for proliferation, but these cells did not contain the proliferation marker BrdU. Additionally, we found that PRA+PRB+ cells in the adult gland lacked expression of phospho-Rb, another protein required for proliferation, but expressed high levels of CDK inhibitors, p21 and  p27 that capable of halting the cell proliferation. From these observations we conclude that it is likely that PRA+ PRB+ cells are cell cycle arrested and do not proliferate. These results imply that if P acts to promote proliferation in the virgin gland through its action in PRA+PRB+ cells it most likely does so through a paracrine mechanism(s). At various developmental stages, especially during pregnancy, a high percentage of cells that expressed only PRB were positive for BrdU. From this observation, we conclude these cells proliferate and that P acting through PRB may directly stimulate proliferation. Our study demonstrates that the rat mammary gland can be a superb animal model to study P action because, similarly to the adult human breast, it expresses PRB and both PRA and PRB are highly co-expressed in the same cell.