Effects of Relatively Low Levels of Mono-(2-Ethylhexyl) Phthalate on Cocultured Sertoli Cells and Gonocytes from Neonatal Rats

Di-(2-ethylhexyl) phthalate (DEHP), one of the abundant man-made environmental chemicals, induces testicular damage in both developing and adult animals. However, the nature and mechanism underlying the action of phthalates on testicular development remain largely unexplored. In the present study, we used cocultures of neonatal Sertoli cells and gonocytes (precursors of spermatogonia) to characterize in detail the effects of mono-(2-ethylhexyl) phthalate (MEHP; the active metabolite of DEHP) on these cells and to explore the underlying mechanism(s). Sertoli cells and gonocytes were isolated from rat pups on the 2nd day after birth, cocultured, and exposed to MEHP at concentrations of 0.01, 0.1, or 1.0 μM, or to 0.5% DMSO (vehicle control), or 10 μM DEHP (negative control) for a total of 48 h. We found that exposure to MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time- and dose-dependent manner. When exposed to 1.0 μM MEHP, many gonocytes started to detach after 12 h of exposure and most gonocytes were lost during the media change at 24 h. Gonocyte detachment was also observed in cocultures treated with 0.1 μM MEHP for 24 h of exposure, but not in cultures treated with 0.01 μM MEHP for 48 h. Detached gonocytes were viable as indicated by their ability to exclude trypan blue. Furthermore, when proliferation of cultured Sertoli cells was detected by BrdU labeling and subsequently quantified, we found that exposure to 0.1 or 1.0 μM MEHP for 48 h resulted in a decrease in labeling indices of 33.6 and 83.6%, respectively, compared to the vehicle control (p< 0.01), while the labeling index was unchanged by treatment with 0.01 μM MEHP. In addition, we also tested the potential effect of MEHP on FSH-stimulated Sertoli cell proliferation by simultaneously treating cultures with 200 ng/ml human FSH and different concentrations of MEHP for 48 h. Exposure to 0.1 or 1.0 μM MEHP resulted in decreases of 24.2 and 74.2%, respectively, in FSH-stimulated Sertoli cell proliferation (p< 0.01). Furthermore, MEHP also inhibited dibutyl cAMP-stimulated Sertoli cell proliferation, regardless of whether dibutyl cAMP was added to the cultures before or at the same time as MEHP. Finally, addition of FSH or dibutyl cAMP had no effect on MEHP-induced gonocyte detachment, and none of the observed effects on either Sertoli cells or gonocytes were detected in control cultures treated with 0.5% DMSO only or with 10 μM DEHP. Therefore, short exposure to low levels