PARENT SESSION

(M730) EFFECTS OF ESTROGEN ON RHESUS MONKEY GRANULOSA CELL PROLIFERATION IN VITRO.

Puttabyatappa, Muraly¹, Fru, Karenne¹, VandeVoort, Catherine², Chaffin, Charles¹, ¹ Medical College of Georgia, Augusta, GA² California National Primate Research Center, Davis, CA

ABSTRACT- Granulosa cells (GC) in the growing follicle of non-human primates express estrogen receptor (ER) , although the high intra-follicular concentrations of estrogen have made it difficult to elucidate a role for this steroid in follicular development. The difficulty in using an ablate / replace paradigm in vivo led us to develop an in vitro culture model with which to address the effects of estrogen on macaque GC proliferation. Rhesus monkeys were treated for 7 days with rec-human FSH and GCs isolated using an ultra-sound guided follicle aspiration technique. The resulting GCs were cultured in the presence of hFSH (control; 25 ng/ml), FSH + insulin/transferrin/selenium (ITS), FSH+ITS+ICI 182,780 (1 M; a synthetic ER antagonist) for 24 hr. ITS increased 3[H]-thymidine incorporation over FSH controls by 3-fold (p<0.05), while ITS+ICI was not different than either FSH or FSH+ITS. The expression of cyclin D2 mRNA increased significantly with ITS treatment (6-fold, p<0.05), as was cdk2 mRNA (18-fold, p<0.05). The mRNA expression of both of these genes was reduced to control levels following ICI treatment. Neither bax nor c-myc mRNA was altered by any treatment, and bcl-2 mRNA was nearly undetectable by real-time RT-PCR. The viability of GCs was determined using an MTT assay and was not reduced by ICI. These results indicate that proliferation of macaque GCs in vitro depends at least in part on locally produced estrogen.

KEY WORDS: granulosa cell, macaque, estrogen, proliferation