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Objectives: The effects of estrogens on immune system formation and function are well documented. Diethylstilbestrol (DES), a synthetic estrogen, has been linked to neoplasia and immune cell dysfunction in humans and animals exposed in-utero. In-vitro effects of DES exposure of murine embryonic stem (ES) cells on the early embryonic immune system development and the expression of cellular surface markers associated with common hemangioblastic and hematopoietic precursors of the endothelial, lymphoid & myeloid lineages were investigated. Hypothesis: Early ES cell expression of CD45 a marker common to lymphoid lineage hematopoietic stem cells and differentiation of lymphoid lineage precursors are affected by in-vitro exposure to DES. Methods: Murine ES cells were cultured using a variety of techniques: an OP9 co-culture system, and formation of embryoid bodies (EBs) in a liquid medium and hanging drop system. The OP9 co-culture system did not appear to give rise to well differentiated lymphoid lineage cells during 12 days of differentiation. The hanging drop EB culture system, previously shown to promote differentiation of endothelial and lymphoid precursor cells, was chosen for further studies of ES cell differentiation. ES cells were harvested at five time points: undifferentiated (day 0), and differentiated (days 3, 8, 12 and 16). Differentiating ES cells were treated with DES beginning on day 3. The synthetic estrogen, DES, was chosen as a treatment because of its similar potency to 17β estradiol and documented association with neoplasia in women exposed in-utero. Surface marker expression, measured by real-time RT-PCR amplification, was recorded using fluorogenic TaqMan(R) probes designed specifically for the surface proteins of interest: oct4, c-Kit, Flk1, ERα, ERβ, CD45, Flt1, & VE-cadherin. Analysis & Results: Changes in surface marker gene expression between day 0 and day 16 of differentiation were analyzed using the RT-PCR threshold counts (CT) and the comparative threshhold cycle method. The expression of each target mRNA was normalized internally to a housekeeping gene (18s rRNA) and calculated relative to day 0. ANOVA (Type 3 fixed-effects analysis, SAS) was performed using the unexponentiated ΔΔCT values. The effects of DES, time, and the interaction between DES and time were evaluated for days 8, 12 and 16. Additionally, the effects of DES on the expression of each marker were evaluated for day 16. Expression of estrogen receptor receptor α & β (ERα & β) in the EBs was established, and did not appear to be affected at any time by treatment with DES. ERα was expressed in significant levels on day 16, while ERβ was expressed in low levels throughout the period of differentiation. The expression of the cell surface marker, c-Kit was significantly (P

For nearly forty years diethylstilbestrol (DES) was administered to pregnant women to maintain healthy pregnancies. During this time, it is estimated that several million men and women have been exposed to DES during sometime of their life. The most common period of exposure was during fetal development. Although rarely used for the maintenance of pregnancy now, its current medical use is restricted to certain clinical situations such as breast and prostate cancer therapies in adults. Thus, DES exposure spans the entire lifetime, from prenatal to geriatric age. Since the early 1950s, health risks were beginning to be associated with prenatal DES treatment. So far only reproductive problems such as infertility, neoplastic diseases of the cervix and vagina and testicular cancers have been well-documented in DES cases. Immunological abnormalities associated with DES are only now beginning to be recognized. Self-reported cases and questionnaire-based studies have revealed increased incidence of infections and autoimmune diseases in DES exposed people. Animal studies that have examined the immunological effects of DES treatment are largely restricted to one gender, or to one dose of DES or to the developmental period. This is an important issue since human exposure to DES occurred in both men and women, at all ages and, at a wide-range of doses. The purpose of these studies was to investigate the immunological consequences resulting from the exposure to DES. Since sensitivity can vary between genders, dose and at the time of exposure, it is critical to investigate the DES-induced immunological changes during all stages of life in both genders. To address these critical gaps in the literature, we examined the immunomodulatory effects of adult and prenatal exposure to DES in males and females. Our findings show that DES effects were evident in both the thymus and spleen. DES markedly affected the apoptosis of thymocytes and the ability of splenic lymphocytes to proliferate in response to stimulants and secrete vital cytokines such as interferon-gamma. Our notable findings were that in-utero exposure to DES resulted in profound alterations in lymphocyte functionality, which were noticed as late as one-year of age. This suggests that alterations to the in utero environment can have deleterious consequences that may be long lasting. These studies have profound implications to the humans and animals exposed to DES, and indirectly to a whole range of other estrogenic compounds.