Prenatal estrogen exposure differentially affects estrogen receptor-associated proteins in rat testis gonocytes

We previously reported that gonocytes from 3-day-old rat testes proliferate in response to estradiol. In the present study, we found that purified gonocytes contained the mRNAs of estrogen receptor beta (ERbeta) and the chaperones Hsp90, p23, and Cyp40, but no inducible Hsp70. Immunoblot analysis showed high levels of ERR, Hsp90, p23, Cyp40, and the constitutive Hsc70 in gonocytes. Prenatal exposure to the estrogenic compounds diethylstilbestrol, bisphenol A, genistein, and coumestrol led to significantly increased Hsp90 mRNA levels in testis, but not p23 and Cyp40. In situ hybridization analysis indicated that Hsp90 mRNA was prominent in gonocytes, where it was increased following phytoestrogen exposure, whereas bisphenol A induced a more generalized increase throughout the testis. Immunoblot analysis of testicular extracts demonstrated that Hsp90 protein levels were significantly increased following estrogen exposure, and immunohistochemical analysis indicated that this increase occurred predominantly in gonocytes. By contrast, no change was observed in the expression of Cyp40, p23, and ERR, whereas Hsc70 was increased by bisphenol A only. Using an antibody and reverse transcriptase-polymerase chain reaction probes specific for Hsp90alpha, we subsequently confirmed that Hsp90alpha was primarily expressed in gonocytes, and that it was increased following estrogen exposure. Hsp90 immunolocalization in fetal and prepubertal testes showed an increased expression in fetal gonocytes upon estrogen exposure, but no difference in the subsets of Hsp90-positive germ cells in prepubertal testes. These results demonstrate that prenatal estrogen exposure specifically affects Hsp90 expression in gonocytes. Considering the interaction of Hsp90 with several signaling molecules, changes in its expression levels may lead to subsequent changes in gonocyte development.