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.

Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.

Vitrification uses cryoprotectants and liquid nitrogen, which may cause osmotic stress and cryodamage to oocytes. Autophagy is widely considered as a survival or responsive mechanism to various environmental and cellular stresses. However, the status of autophagy in vitrified-warmed oocytes has not been studied. In this work, we investigated if vitrification-warming process induces autophagy in mouse oocytes. Four-week-old female ICR mice and GFP-LC3 transgenic mice were used. The mice were superovulated with 5IU PMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts. Oocytes obtained from several mice were pooled and divided into three groups. Group1: fresh oocytes. Group2: oocytes treated with vitification solutions (1.3 M EG+1.1 M DMSO and 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 2.5 min) and warming solutions (0.5 M, 0.25, 0,125, and 0 M sucrose at intervals 2.5 min). Group3: vitrified-warmed oocytes (loaded onto an EM copper grid, and were stored in LN2 for 2 weeks). RT-PCR and confocal live imaging of GFP-LC3 were performed to examine the effects of vitrification-warming process on autophagy in oocytes. In RT-PCR analyses, expression of autophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, and Beclin1 was examined. Expression of Atg7 and Atg12 was slightly reduced in Group 3 (vitrified-warmed oocytes). The expression levels of other Atg genes did not change. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that some vitrified-warmed oocytes showed green puncta which indicate autophagic activation. All oocytes of Group 1 and Group 2 show no puncta formation. Our results suggest that induction of autophagy may serve as an indicator of conditions of vitrification-warming process. Moreover, it offers the possibility that development of methods to modulate autophagic response during cryopreservation could improve efficacy of oocyte cryopreservation.

Autophagy is a major cellular catabolic pathway and is tightly associated with survival and death of cells. The involvement of autophagy during prolonged survival of blastocysts in the uterus is established and it was assumed that ovarian steroid hormones – estrogen (E2) and progesterone (P4) – play important roles in its regulation. The uterus is a major target organ of E2 and P4. To examine if E2 or P4 modulate autophagy in the mouse uterus in vivo, the following three systems were used. 1) Normal pregnancy model (days 1 to 8); 2) delayed implantation model; 3) ovariectomized (OVX) mice model treated with single steroid hormone. Six-week-old virgin ICR mice were used for pregnancy and OXV. OVX mice received P4 (1 mg/0.1 ml) or E2 (100 ng/0.1 ml) after 12 days of rest. Collected uteri were subjected to Western blotting and immunofluorescence staining using anti-LC3B antibody to monitor autophagy. In pregnant mouse uterus, the autophagic response was downregulated after implantation. In OVX model, either E2 or P4 injection downregulated the autophagic response in the uterus within several hours. To confirm whether hormone-induced downregulation is mediated by classical estrogen receptor (ER) and progesterone receptor (PR), receptor antagonists (ICI 182,780 and RU-486) were co-treated. Antagonist-treated uteri showed recovery of autophagic response, suggesting that ER or PR mediates hormonal effects on autophagy. In oder to determine which signaling pathway is involved in autophagic regulation by E2, rapamycin (5 mg/kg), a mTOR inhibitor, and LY294002 (5 mg/kg), a PI3 kinase inhibitor, were used. Rapamycin and LY294002 were injected just before E2 injection to OVX mice. Western blotting was performed by using anti-phospho-mTOR and anti-AKT antibodies. We observed that rapamycin treatment partially antagonized downregulation of autophagic activation by E2, whereas LY294002 treatment did not have any effect. Therefore, downregulation of autophagy by E2 seems to be partially mediated by mTOR pathway. Collectively, this study suggests that ovarian steroid hormones are upstream controllers of autophagic response in the mouse uterus.

The anandamide signaling plays various roles in directing reproductive processes. Mouse embryos are shown to express high levels of CB1 receptor (CB1R). It has recently been shown that an analog of anandamide induces autophagy-mediated cell death through stimulation of ER stress response in glioma cells. Since adverse effects of high levels of anandamide agonists on embryo development and implantation are well known, we hypothesized that anandamide mediates an autophagic response in embryonic cells as in cancer cells via highly abundant CB1R on embryos. We tested this hypothesis by using a stable anandamide agonist, Methanandamide (MET) in three embryonic cell systems, i.e., mouse embryonic fibroblasts (MEF), trophoblast stem (TS) cells, and preimplantation embryos from mice. RT-PCR, immunofluorescence staining, and Western blot analysis were used to examine the effects of anandamide on autophagy in these systems. In MEF cells, the conversion of LCI to LCII was heightened by methanandamide (MET), and AM251, a selective CB1 antagonist partially reversed the effects of MET. Treating MEF cells with a high level of MET induces clustering of GFP-LC3, seen as large puncta throughout the cytoplasm. At 28 nM concentration, MET also weakly increased LC3II in TS cells. When MET was injected to day 4 pregnant mice, autophagy was increased in blastocysts in utero as demonstrated by the increased number of LC3 puncta. Formation of numerous autophagic vacuoles was also confirmed by electron microscopic observation. In conclusion, this work suggests that the anandamide-CB1 signaling pathway may be one inducer of autophagy in embryonic cells.