The present study indicated that severe changes in Ocl morphology are associated with the cells' inability to carry out resorption in the osteosclerotic estradiol-treated mouse. Although osteoclasts retained TRAP activity, ultrastructural alterations were identified in estradiol-treated animals in both the sealing patterns of osteoclasts against the bone surface and in altered brush border morphology. Proper sealing of the osteoclast against the bone surface and a functional brush border are two ultrastructural components believed to be of major importance in Ocl function. The ultrastructural alterations identified in estrogen-treated mice were very likely the cellular functional basis for the decreased resorption and development of osteosclerosis in estrogen-treated mice. Osteoclast function and ultrastructural morphology have been shown to be highly responsive to anti-resorbing agents such as ethane-1-hydroxy-1,1-diphosphonate (EHDP) [8, 9], dichloromethylene diphosphanate (Cl2MDP) , calcitonin [11,12,13], other anti-resorptive agents, such as mithramycin , and to gallium nitrate .
Although histochemical TRAP localization provides an accurate and sensitive histologic method for identification of Ocl, understanding osteoclastogenesis and Ocl function remain a challenging area of bone research. The current understanding of Ocl function is that the Ocl seals its perimeter along the bone surface by means of the podosome [16, 17, 18] . Within this sealed margin, projections from the cytoplasm form the ruffled border, a specialized Ocl-extracellular space in which bone resorption occurs. In the present study in the mouse, differentiation of osteoclast progenitors appears to be dependent on the direct interaction of bone marrow stromal cells with Ocl precursor cells [19, 20]. Recent data of Baylink  and Linkhart  provide evidence that two inbred strains of mice with different bone densities may have fundamental differences in bone resorption (not bone formation), and that genes affecting the bone marrow Ocl precursor population may contribute to relative differences in the C57BL/6J and C3H/HeJ strains. In vivo and in vitro studies of avian osteoclasts led Pederson et al to suggest that modulation of the Ocl response to estrogen may be controlled by alterations in the Ocl estrogen receptor level .
Maturation of osteoclasts from their macrophage precursors requires marrow stromal cells or their osteoblast progeny as shown by Udagawa et al . These cells produce macrophage colony-stimulating factor (M-CSF) and the receptor for activation of nuclear factor kappa b (NF-kB) (RANK) ligand (RANKL). M-CSF is essential for macrophage maturation, but formation of osteoclasts also requires contact between osteoclast precursors and stromal cells or osteoblasts . As described by Hofbauer et al , the quantity of bone resorbed depends on the balance between expression of RANKL and of its inhibitor, osteoprotegerin (OPG). Hofbauer et al suggest that the stimulation of the pool of M-CSF precursors to committed osteoclastogenesis by RANKL may be one of the central pathophysiologic pathways involved in increasing the number of osteoclasts in osteoporosis.
Futures studies of the estrogen-treated mouse model are needed which are designed to address osteoclastogenesis and osteoprotegerin (OPG) secretion by osteoblasts. OPG is currently hypothesized to be an important paracrine mediator of the antiresorptive action of estrogen on bone cells . In osteoblast cell lines exposed to estrogen, OPG mRNA and protein levels were increased compared to controls. Hofbauer et al have suggested that estrogen may exert its anti-resorptive effects by enhancement of OPG secretion by osteoblasts .
Future studies should also assess the temporal development of functional changes in the osteoclast populations during estrogen treatment, and the patterns of osteoclast death with special attention to apoptosis. Estradiol has been shown to modulate IL-1 action on human osteoclasts in vitro; estrogen administration inhibited IL-1-mediated cytokine (IL-8) mRNA induction and caused increased Ocl apoptosis . When avian Ocl were exposed to estradiol in vitro, the Ocl plasma membrane became depolarized and there was a marked decrease in potential, suggesting that estrogen may regulate osteoclasts through ion channel activities . Estradiol has also been shown to inhibit acid production in avian Ocl . The human pre-Ocl cell line FLG 29.1 has specific binding sites to 17β-estradiol on the cell surface, and estradiol exposure increases the cellular pH, cAMP, cGMP and intracellular calcium .
In vivo work by Jilka et al has shown that Il-6 can prevent the bone loss associated with ovariectomy in the mouse model . Girasole et al have recently shown that production of IL-6 is inhibited in vitro by exposure of marrow cells to 17β-estradiol; cultures of mouse bone cells showed suppressed osteoclast development when exposed to either 17β-estradiol or a neutralizing antibody to Il-6 . Future in vivo studies combining estradiol and selected interleukins may further elucidate how the network of signals from the marrow microenvironment influences both Ocl development and Ocl function.