In this study, we have examined the influence of diethylstilbestrol (DES) a
nd 17 beta -estradiol on the proliferation of TM3 Leydig cells, a normalize
d mouse cell line. Cells were treated with seven different concentrations (
1 pg-1 mug/ml) of DES or 17 beta -estradiol, and cell growth was measured a
t 24-, 48-, and 72-h periods. DES treatment resulted in a significant (p <
0.05) stimulation of cell proliferation. We observed two independent peaks
of cell proliferation, one at 1 pg/ml DES (186.87%) and the other at 100 ng
/ml DES (248.23%). Cytotoxicity was noted at all time periods with 1 <mu>g/
ml DES treatment. 17 beta -estradiol treatment resulted in a significant st
imulation of cell proliferation (p < 0.05) with a trend similar in response
to that of DES treatment, as peak proliferation was noted with 1 pg/ml 17<
beta>-estradiol (125.27%) and 10 ng/ml 17 beta -estradiol (138.31%). Based
on these data, it appears that DES is more mitogenic in these Leydig cells
compared to 17 beta -estradiol. Furthermore, for the first time, we detecte
d that both DES and 17 beta -estradiol were able to stimulate proliferation
of Leydig cells in a biphasic fashion. Cell cycle kinetic analysis reveale
d that cell entry into the S-phase was higher in the DES treated cells comp
ared to the controls, and doubling times of DES exposed cells were signific
antly reduced (p < 0.05). Go-administration of tamoxifen at a concentration
1000-fold higher than either DES or 17<beta>-estradiol resulted in complet
e inhibition of cell proliferation. Analysis of expression of ER alpha and
ER beta by RT-PCR in untreated Leydig cells, as well as Leydig cells expose
d to 1 pg/ml DES, revealed that the transcripts of ER alpha and ER beta wer
e not detectable even after 40 cycles of amplification. A 100-ng/ml dose of
DES induced ER alpha expression by 20-fold. These data suggest that estrog
en exposure-mediated increases in cell proliferation, coupled with the decr
ease in cell cycle time, may allow greater accumulation of DNA damage to oc
cur in the testicular target cells compared to untreated cells under normal
cell cycle control. In addition, an unidentified estrogen receptor may be
responsible for the mitogenic activity of estrogens at low levels.