DETECTION OF ESTROGEN-RECEPTOR-ALPHA, CARBONIC-ANHYDRASE-II AND TARTRATE-RESISTANT ACID-PHOSPHATASE MESSENGER-RNA IN PUTATIVE MONONUCLEAR OSTEOCLAST PRECURSOR CELLS OF NEONATAL RATS BY FLUORESCENCE IN-SITU HYBRIDIZATION

Increasing evidence suggests that estrogen deficiency in women promote s the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and loca l factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP ) and carbonic anhydrase II (CA II), characterizes osteoclast differen tiation. To evaluate the possibility that estrogen may have a direct e ffect on osteoclast precursor cells, we investigated the mRNA levels o f estrogen receptor alpha (ER alpha), TRACP and CA II genes in neonata l rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display str ongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions simil ar to those observed in osteoclasts. Both CA II and TRACP mRNAs were d etected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To d etermine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in t heir cytoplasm. In contrast, only a few multinuclear osteoclasts in th e bone imprints possessed ER gene transcripts. Interestingly, the leve ls of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybrid ization resulted in a significant loss of signal in these cells. The r esults of these studies suggest that estrogen may have a direct role i n modulating the recruitment of osteoclast precursor cells during oste oclastogenesis.