We have investigated the capacity of vaginal and uterine epithelia (VE and UE) to reexpress normal morphology and hormone responsiveness following cell culture. VE and UE from adult ovariectomized mice were grown in a collagen gel matrix with serum-free medium for 7-10 days. Proliferation of these cells occurs in the absence of 1713-estradiol and is not stimulated by 17J3-estradiol; the VE usually does not keratinize or stratify in vitro. Cultured VE and UE were recombined with homologous vaginal or uterine stroma (VS and US, respectively) and these recombinants were grown under the renal capsule of female hosts for 4 weeks. The epithelium of the VS + VE recombinants cycled, proliferated and stratified in response to estrogen and mucified normally in response to progesterone. The UE that was grown in vivo with cultured US also showed normal morphology and estrogen responsiveness. These changes in the UE and VE were not simply a result of return to the in vivo environment, as epithelia alone in collagen gels transplanted under the renal capsule did not survive. These results indicate that both VE and UE, which are not estrogen-dependent in vitro, reexpress their estrogen dependency and normal morphology when recombined with homologous stroma and grown in vivo. Thus, the changes these cells show when grown in culture are the result of altered conditions in vitro rather than an irreversible alteration in the cells themselves or the selection of specific subpopulations that are not nmitogenically or morphologically responsive to estrogen under any condition.Cell culture has provided a powerful tool for studying many aspects of cell growth and function under controlled conditions. However, the utility of culture systems has been limited because of discrepancies in cell behavior in vitro and in vivo and the possibility that cells may lose their identity and capacity for normal function in vitro. Epithelial cells in culture exhibit marked changes in shape, protein-synthetic patterns, hormone responsiveness, and function compared with intact epithelia in vivo (1-3). Paradoxically, hormones that are primary regulators of cellular proliferation in vivo may have no growth-promoting effect on their target cells in vitro. This phenomenon is illustrated by the repeated observations that estrogen, the principal mitogen for female reproductive tract epithelium in vivo (4), is not mitogenic for isolated reproductive tract (or mammary gland) epithelia in vitro (5-10). For example, vaginal epithelial (VE) and uterine epithelial (UE) cultures from adult ovariectomized (Ovx) mice exhibited 4-to 8-fold and 3-fold increases, respectively, in DNA content when cultured for 10 days in collagen gel with serum-free medium in the absence of estrogen (5, 8). The addition of estrogen to the medium did not stimulate proliferation of these cells and, at higher concentrations, was actually inhibitory (5,8). In addition, the VE did not usually stratify or keratinize in vitro.The lack of estrogen mitogenicity in vitro raises the possibilit...

Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrgl mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4-to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. (3, 4) and mouse vaginal epithelial outgrowths (2) failed to cornify in response to estradiol, whereas others have reported that the addition of estrogen to the culture medium evoked cornification of vaginal explants from rats (5-8) and mice (9-13). Martin (14) described growth and cornification of cultured mouse vaginal epithelium in the absence of estrogen. The possible role of the stroma and the difficulty of accurate quantification of increases in cell number are among the problems with both organ and cell culture methods. Furthermore, in organ culture, tissues degenerate with time; in cell culture, lack of sustained cell division and decreasing viability occur with time (2). To examine the possible direct effect of estrogen on proliferation and cornification of vaginal epithelium, a system is needed for the in vitro culture of isolated epithelial cells.Recently, a collagen gel culture system was developed for mammary epithelial cells that results in sustained growth of mouse, rat, and human cells in primary culture (15)(16)(17)(18)(19)(20)(21). This in vitro system has been used successfully in our studies for the growth of mouse vaginal epithelial cells, as judged by an increase in cell number, and for the possible hormonal regulation of differentiation of these cells in the resulting cultures. In the present communication, we report on the sustained growth of cultured vaginal epithelial cells in a serum-free medium and on the failure of estrogen to stimulate further growth.MATERIALS AND METHODS Epithelial Cell Isolation. Normal vaginae dissected from 50-to 60-day-old BALB/cCrgl mice 6-7 days after ovariectomy were transversely sectioned and then incubated in Hank's balanced salt solution containing 0.1% collagenase (CLS III, 175-184 units per mg; Worthington) and S mg of bovine serum albumin fraction V per ml for 2 hr at 370C in a shaking water bath. Epithelial sheets manually separated from stroma were minced into small clumps with a razor blade on a Teflon block. Cell clumps were collected by centrifugation at 1,000 x g for 5 min.More than 85% of the cells were found to be viable as determined by the trypan blue exclusion test. Cell number was estimated by mixing 1 vol of cell suspension with 9 vol of 0.02% crystal violet in 0.1 M citric acid and counting stained nuclei in a hemocytometer.Preparation of Collagen Gels. Collagen solution and gels were prepared as originally described (22). Briefly, 1 g of rat ta...

Amino 1H NMR line width as a measure of amino proton exchange in guanosine compounds is completely unaffected by the addition of ca. 1 M tris(hydroxymethyl)-aminomethane, imidazole, 2-(N-morpholino)ethanesulfonic acid, glycine, or cacodylate, all shown to be effective buffer catalysts in adenosine and cytidine proton exchange. Line broadening, seen only with phosphate and acetate, is established by intermolecular interactions, as well as by amino to water proton exchange. This absence of buffer catalysis of exchange is accounted for by the relatively small implied effect of G(N-7) protonation on amino acidity, based on similar observations with 7-methylguanosine as a model for endocyclic protonation. The requirement for diffusion-controlled proton transfer in buffer catalysis is achieved by nucleobase protonation in adenine and cytosine, but not in guanine.

The adult mouse uterus is composed of luminal epithelium, endometrial connective tissue into which glands project, and a fibromuscular wall. Analyses made on the whole organ do not provide information on functional differences among the several component cell populations, which may show different hormone sensitivities.' Therefore, attempts have been made to obtain homogeneous preparations of epithelial and stromal cells from human,2>°3> rabbit4),5) and rat6~,7r uteri. We present here a procedure for the isolation of normal mouse luminal' endometrial epithelial cells and describe their growth in serum-free media.Materials and methods. Normal uteri dissected from ca. 35-day-old BALB/ cCrgl mice 5-7 days after ovariectomy were transversely sectioned (ca. 5 mm length) and then incubated in Hanks' balanced salt solution (HBSS, GIBCO) containing 1% trypsin (1: 250, Difco Lab., Detroit, MI) for 30 min at 37°C in a shaking water bath.After incubation, bovine serum albumin fraction V (5 mg/ml) (BSA, Sigma Chemical, St. Louis, MO) was added to the dissociation flask for 5 min at room temperature to inactivate the trypsin. Uterine segments were transferred to fresh HBSS and the luminal epithelium was separated from the glands and fibromuscular wall by gently squeezing along the length of the uterine tube with jeweller's forceps. The epithelium expressed as tubes or sheets was minced into ca. 1-mm3 clumps with a razor blade on a teflon block. Cell clumps were collected by centrifugation at 1,000 X g for 5 min. More than 80% of the cells were found to be viable as determined by the trypan blue exclusion test. Collagen solution and gels were prepared as originally described.8) Briefly, cells were added to the cold mixture; 0.5 ml, containing 0.5-4.S x 105 cells, was layered onto a 0.3-m1 bed of gelled collagen and allowed to gel at room temperature in each well of Falcon multiwell plates. Cells were also seeded on the gelled collagen. The cells were cultured in a serum-free complete (SFc) medium consisting of Dulbecco's modified Eagle medium/Ham's F-12 medium (D : H, GIBCO), 1:1 (vol/vol), containing NaHCO3 at 1.2 g/liter with penicillin (50 U/ml) and streptomycin (50 µg/ml). The D : H medium was supplemented with insulin (10 µg/ml), transf errin (10 µg/ml), cholera toxin (10 ng/ml), and BSA (5 mg/ ml), all from Sigma Chemical, and with epidermal growth factor (E GF,10 ng/ml, Collaborative Research, Waltham, MA). For a suboptimal serum-free medium, only insulin (10 µg/ml) and BSA (5 mg/ml) were added. Cultures were incubated at 37°C in humidified 95% air-5% CO2 mixture. The medium was changed every two days.

No abstract