Effects of estrogen status in osteocyte autophagy and its relation to osteocyte viability in alveolar process of ovariectomized rats

Abstract

Estrogen maintains osteocyte viability, whereas its deficiency induces osteocyte apoptosis. As autophagy is important for osteocyte viability, we hypothesized whether the anti-apoptotic effect of estrogen is related to autophagy in osteocytes. Thirty adult female rats were sham-operated (SHAM) or ovariectomized (OVX). After three weeks, twelve rats of SHAM and OVX groups were killed before treatment (basal period), whereas the remaining rats received estrogen (OVXE) or vehicle (OVX) for 45 days. Fragments of maxilla containing alveolar process of the first molars were embedded in paraffin or Araldite. Paraffin-sections were stained with hematoxylin/eosin for histomorphometry, or subjected to the silver impregnation method for morphological analysis of osteocyte cytoplasmic processes. Autophagy was analyzed by immunohistochemical detections of beclin-1, MAP-LC3α and p62, whereas apoptosis was evaluated by immunohistochemical detections of cleaved caspase-3 and BAX, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method and by ultrastructural analysis. Araldite-semithin sections were subjected to the Sudan-black method for detection of lipids. OVX-basal group showed high frequency of caspase-3-, TUNEL- and p62-positive osteocytes accompanied with low frequency of beclin-1- and MAP-LC3α-positive osteocytes. At 45 days, OVXE group exhibited higher number of osteocytes, higher frequency of beclin-1- and MAP-LC3α-positive osteocytes, and lower frequency of caspase-3, BAX-, TUNEL- and p62-positive osteocytes than OVX group. Significant reduction in bone area was observed in the OVX compared to OVXE and SHAM groups. The highest frequency of Sudan-Black-positive osteocytes and osteocytes with scarce cytoplasmic processes, or showing apoptotic features were mainly observed in OVX groups. Our results indicate that estrogen deficiency decreases autophagy and increases apoptosis, whereas estrogen replacement enhances osteocyte viability by inhibiting apoptosis and maintaining autophagy in alveolar process osteocytes. These results suggest that the anti-apoptotic effect of estrogen may be, at least in part, related to autophagy regulation in osteocytes.