Estrogen treatment reduces Ig gene rearrangements in bone marrow.

(A) DNA samples representing equivalent numbers of bone marrow lymphocytes from control and E2-treated mice were evaluated for D_H-J_H and V_H-DJ_H gene rearrangements by PCR and specific products were labeled with respect to rearrangements to J1, J2, or J3. D_H-J_Hrearrangements were detected with a primer that recognizes 9 of 10 known D minigenes and a J3 primer.24V_H-DJ_H rearrangements were detected using primers specific for the Q52 V region family and a J3 primer.3,24 Genomic DNA was serially diluted 1:1,1:10,1:100 for semiquantative analysis and 25 cycles of amplification was performed. Alpha actin was used as a control for genome representation. Densitometry confirmed that equal amounts of DNA were compared. These data are representative of results obtained in 3 independent experiments. (B) CD45R⁺CD43⁻sIg⁻ B-lineage precursors in estrogen-treated mice are enriched for cells with germline Ig genes. CD45R⁺CD43⁻ sIg⁻ cells were sorted from control or estrogen-treated mice. PCR was performed with genomic DNA, using primers that amplify segments normally deleted during Ig gene rearrangement (see “Materials and Methods”). DNA was amplified for 30 cycles and alpha actin was used as a control for genome representation. These data were obtained by pooling cells from 4 control and 4 estrogen-treated mice. RAG-1^−/−bone marrow was used as a control for germline DNA. Cells sorted from control and estrogen-treated mice were 99% and 100% pure, respectively, based on postsort analyses.