(A) A schematic diagram of ex vivo bone marrow osteoclast differentiation. (B) Expression of Nur77, Nurr1, and Nor-1 encoding genes (Nr4a1, Nr4a1, Nr4a3) during a time course of RANKL-induced osteoclast differentiation (n = 3 mice). Nfatc1 mRNA level was similar to Nr4a1 mRNA level on day 2–3. (C–D) Osteoclast differentiation was enhanced in Nur77 KO cultures compared to WT control cultures. (C) Expression of osteoclast differentiation markers on day 3 (n = 4 mice). (D) Representative images of the TRAP-stained osteoclast differentiation cultures. Mature osteoclasts were identified as multinucleated TRAP+ (purple) cells on day 9. Scale bar, 25 μm. Quantification of osteoclast size, osteoclast number per area, and osteoclast-resorptive activity by calcium release from bone plate to culture medium is shown (n = 4 mice in triplicate cultures). Oc, osteoclast. (E) Osteoclast apoptosis was unaltered, quantified by the expression of apoptosis genes on day 9 of osteoclast differentiation cultures (n = 4). (F) Osteoblast differentiation was unaltered in Nur77 KO cultures, measured by the expression of osteoblast markers (n = 4). (G–O) Nur77 KO mice exhibited bone loss. Tibiae from Nur77 KO mice or WT littermate controls (3 month old, male, n = 6) were analyzed by μCT. (G) Representative images of the trabecular bone of the tibial metaphysis (top) (scale bar, 10 μm) and the entire proximal tibia (bottom) (scale bar, 1 mm). (H–O) Quantification of trabecular bone volume and architecture. (H) BV/TV, bone volume/tissue volume ratio. (I) BS, bone surface. (J) BS/BV, bone surface/bone volume ratio. (K) Tb.N, trabecular number. (L) Tb.Th, trabecular thickness. (M) Tb.Sp, trabecular separation. (N) Conn.D., connectivity density. (O) SMI, structure model index. (P) Serum CTX-1 bone resorption marker was increased (3 month old, male, n = 6). (Q) Serum P1NP bone formation marker was unaltered (3 month old, male, n = 6). (R–S) Bone histomorphometry (3-month-old, male, n = 6). (R) Quantification of osteoclast surface (Oc.S/B.S) and osteoclast number (Oc.N/B.Ar). (S) Quantification of osteoblast surface (Ob.S/B.S) and osteoblast number (Ob.N/B.Ar). B.S, bone surface; B.Ar, bone area. Error bars, SD.