Estrogen deficiency induces bone loss by increasing bone resorption through upregulation of receptor activator of nuclear factor κB ligand (RANKL). RANKL is secreted by osteoblasts, but more recently also bone marrow (pre)-adipocytes have been shown to express RANKL. Estrogen deficiency increases bone marrow adipose tissue (BMAT) and higher BMAT is associated with postmenopausal osteoporosis. Therefore, the aim of this study was to determine the protein expression of RANKL in both bone marrow adipocytes and osteoblasts in C3H/HeJ mice after ovariectomy or sham surgery.

14 weeks old, female C3H/HeJ mice (n=20) were randomized to sham surgery (Sham) or ovariectomy (OVX). After 4 weeks animals were euthanized. BMAT volume fraction (Ad.V/Ma.V) was quantified in the proximal metaphysis of the right tibia by polyoxometalate-based contrast enhanced nanoCT. Left femora were decalcified, embedded in paraffin and immunohistochemically stained for RANKL. The percentage of RANKL positive bone marrow adipocytes (RANKL.Pos.N.Ad/N.Ad) and the percentage of RANKL positive osteoblasts covering the bone surface (RANKL.Pos.B.Pm/B.Pm) were measured. The effects of OVX were analyzed by Student’s t-test or Mann-Whitney U test, depending on the distribution of the data.

RANKL was detected in osteoblasts, osteocytes and bone marrow adipocytes. We found that OVX significantly increased BMAT volume fraction (median Ad.V/Ma.V (IQR): Sham: 1.4 (4.9)%; OVX: 7.2 (7.3)%: p=0.008), the percentage of RANKL positive bone marrow adipocytes (mean RANKL.Pos.N.Ad/N.Ad ±SD: Sham: 42±18%; OVX: 62±14%; p=0.039) and the percentage of RANKL positive osteoblasts covering the bone surface (mean RANKL.Pos.B.Pm/B.Pm ±SD: Sham: 27±7%; OVX: 40±11%; p=0.018) compared to sham surgery.

Ovariectomy increased the percentage of RANKL positive bone marrow adipocytes and increased the percentage of bone surface covered with RANKL positive osteoblasts in C3H/HeJ mice. As not only the percentage of RANKL positive bone marrow adipocytes increased after ovariectomy but also the total quantity of bone marrow adipose tissue, RANKL produced by bone marrow adipocytes might contribute to ovariectomy induced bone loss in C3H/HeJ mice.