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Mouse vaginal epithelial cells were isolated from intact 21-day-old BALB/cCrgl mice and cultured in a serum-free medium (SF20: basal medium supplemented with insulin, epidermal growth factor, transferrin, and bovine serum albumin--fraction V) to examine the proliferation, differentiation, and specificity of estrogen-induced growth retardation in vitro. Histologic and ultrastructural studies showed that vaginal epithelial cells undergo differentiative changes in vitro in the absence of estrogen, and that these changes are similar to those induced in vivo by estrogen. Addition of 17 beta-estradiol inhibited cellular proliferation in a dose-dependent manner. Whereas other estrane derivatives (17 alpha-estradiol and estriol) also significantly retarded cellular proliferation, cholesterol, testosterone, and progesterone had no effect. Keoxifene, an antiestrogen, significantly reversed estrogen-induced growth inhibition, resulting in proliferation of estrogen-treated cells equivalent to that of the untreated control. The results suggest that both proliferation and differentiation of prepubertal mouse vaginal epithelial cells in vitro are estrogen-independent, and that the growth inhibition is a specific estrogen-induced response.

Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel matrix and a serum-free medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17 beta-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen.