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The effect of Atg7 deletion on the uterine autophagy
Autophagy is a self-degradative process which accompanies the formation of double-membraned vesicles inside the cell. In the mouse uterus, autophagy is enhanced during steroid hormone deprivation and associated with acute inflammation. There are 17 major Autophagy related genes (Atg). Herein we investigated the role for Atg7 by using uterine cell-specific deletion model of this gene. We crossed Atg7flox/flox (Atg7f/f) mouse and Anti-Mullerian hormone type 2 receptor (Amhr2)-Cre mice (Amhr2-Cre; Atg7f/f). Amhr2 is mainly expressed in stroma and myometrium in the uterus, ovary, and oviduct, during 30 to 60 days. To confirm the region of Cre expression and to monitor whether conditional deletion of Atg7 was by Cre recombinase, we isolated uterine epithelial and stromal cells from 8 and 16 weeks mice by enzymatic digestion and performed RT-PCR. We confirmed that Amhr2-Cre is expressed in stoma and myomotrium, but not in epithelium. Then we examined the uterine histology and embryonic development of day 3 pregnant Amhr2-Cre; Atg7f/f mice. However, there was no specific difference between Atg7f/f (control) and Amhr2-Cre; Atg7f/f mice. To examine the effect of hormone deprivation, we performed western blotting and immunofluorescence staining of p62 (SQSTM1), an indicator of autophagic flux, and LC3B, a marker of autophagic activation, in Amhr2-Cre; Atg7f/f mice ovariectomized (OVX) for 2 weeks. p62 increased dramatically in OVX Amhr2-Cre; Atg7f/f uteri but not in control mice, suggesting that autophagic activation did not occur in the absence of Atg7 in the uterine stroma and that this led to massive accumulation of p62 in this cell type. p62 marks to-be-degraded proteins and target them for autophagic-lysosomal degradation. Thus it is predictable that Atg7-driven uterine autophagy is responsible for degradation of macromolecules during hormone deprivation.
