ESTROGEN-RECEPTORS ARE PRESENT IN HUMAN GRANULOSA-CELLS

Recent studies failed to detect estrogen receptors in primate follicle s. This study was initiated to determine whether estrogen receptor (ER ) messenger ribonucleic acid (mRNA) is present in human granulosa cell s and, further, if functional ER proteins are present. To evaluate the presence of ER, RNA from human granulosa cells obtained at the time o f oocyte retrieval for assisted reproduction was extracted, and comple mentary DNA synthesis was performed by the reverse transcriptase-polym erase chain reaction. Oligonucleotide primers were used to amplify bas epairs 570-852 in the B- and C-domains of the ER mRNA. Southern blotti ng was performed and confirmed that the amplified DNA fragment identif ied in granulosa cells represented ER. By reverse transcriptase-polyme rase chain reaction, mRNA for the ER is clearly identified in primary human granulosa cells obtained at the time of oocyte retrieval. To exp and these studies and determine whether functional ER were present in human granulosa cells in culture, a simian virus-40-transformed human granulosa cell line was studied. Cells were transfected with a plasmid containing an estrogen response element up-stream from the bacterial reporter gene chloramphenicol acetyltransferase (CAT). In transfected cells, CAT activity is inducible by estradiol if endogenous functional ER are present. In these studies, the transfection analysis confirmed that functional, transcriptionally competent ER are present in a huma n granulosa cell line, with a 4- to 5-fold enhancement of CAT activity demonstrated after the addition of estradiol compared to that in nonh ormone-treated cells. In conclusion, ER mRNA is present in human granu losa cells. Functional ER are also demonstrated in a transformed human granulosa cell line. We hypothesize that low, but biologically signif icant, amounts of ER protein are present in human granulosa cells, whi ch are not routinely detectable by standard assays.