1. Autophagy and Apoptosis are induced in the human endometrium during the menstrual cycle
We investigated whether autophagy is induced during the menstrual cycle in the human endometrium from ovarian endometriotic cysts. We measured the levels of microtubule associated protein 1 light chain 3 alpha (LC3), microtubule associated protein 1 light chain 3 beta phosphatidylethanolamine conjugate (LC3B-II), and Beclin 1. LC3B-II localizes to autophagosomes and is one of the best-characterized markers of autophagy. Beclin 1 is an essential protein for the initiation of autophagosome formation. As autophagy marker protein [11-12], LC3 was expressed in glandular and stromal cells throughout the menstrual cycle and was localized within the cytoplasm. As shown in Fig 1, in the early (Fig. 1A) and late proliferative phases (Fig, 1B), LC3B-II was negative or very weakly positive. However, the levels of LC3B-II in the secretory endometrium was significantly higher than that in the proliferative endometrium, peaking during the late secretory phase (Fig. 1D). Similarly, the beclin-1 level also increased significantly during the secretory phase (Fig. 1 G,H). Subsequently, we also evaluated the level of the apoptosis marker cleaved caspase-3 . Endometrial cell autophagy is associated with apoptosis during the menstrual cycle, and the level of cleaved caspase-3 also increased significantly during the secretory phase (Fig. 1K,L).
Both western blotting (Fig. 1M) and semi-quantitative western blotting analysis (Fig. 1N-P) also revealed that autophagy and apoptosis were upregulated in the human endometrium during the secretory phase.
2. Autophagy-related factors are decreased in obese patients during the secretory phase
We investigated whether autophagy was upregulated during the secretory phase in obese patients. On the same gel, we compared the levels of LC3B-II and Beclin 1 directly at the early proliferative phase and the late secretory phase in the endometria of lean and obese patients. In the obese patients, as shown in Fig. 2 D and H, immunofluorescent staining showed that LC3B-II and Beclin 1 levels were lower than those in lean patients during the late secretory phase (Fig. 2 C and G). Western blotting (Fig. 2I,K) and semi-quantitative (Fig. 2J,L) western blotting analysis also revealed that autophagy might be impaired in obese patients during the menstrual cycle.
3. Downregulation of autophagy-related genes expression in endometrial tissue during the late secretory phase from obesity patients
Autophagy-related (ATG) proteins are eukaryotic factors involved in autophagosome formation and in various stages of the autophagic process. Autophagy is initiated by post-translational modifications of ATG proteins. FOXO1 is involved in activation of various transcription factors, the regulation of cell cycle, apoptosis, autophagy. We determined the expression levels of mRNA encoding 3 important autophagy-related proteins, ATG5, ATG16L1, ATG7, as well as transcription factor forkhead box protein O1 (FOXO1) in the endometrium during the late secretory phase from obesity patients and control subjects. Research demonstrated that these proteins have a positive correlation with endometrium decidualization [14-16]. As shown in Fig. 3, the expression levels of ATG5, ATG16L1 and FOXO1 were significantly decreased in the endometrial tissue of from obesity patients. The expression levels of ATG7 did not significantly differ between the two groups, however, there was a trend towards a decrease in obesity patients. We demonstrate that the downregulation of autophagy-related gene expression lead to altered autophagy in obesity patients.
4. Autophagy-related Inflammatory cytokines are increased in the endometrium of obese patients
There is considerable evidence that defective autophagy induces inflammation: ATG5 deficiency stimulated an increase in tumor necrosis factor alpha (TNF-α) production ; high amounts of the proinflammatory cytokine Interleukin (IL)-1β and IL-18 were released from Atg16L1-deficient macrophage ; elevations in IL-1β were also observed in the ATG7 deficient macrophages . Besides, ATG5 is also involved in the immune system, regulating innate and adaptive immune responses, including the activation of interferon (IFN)-I [25-26]. Therefore the expression levels of above related inflammatory cytokines in the endometria of lean and obese patients were assessed. We observed marked increases in the mRNA expression levels of inflammatory-related factors, such as TNF-α, IL-1β, IL-18 and IFN -I, monocyte chemoattractant protein-1 (MCP-1), and accompanied by a significant decrease in the IL10 mRNA level. As shown in Fig. 4, IL -1b, MCP-, and TNF- a as pro-inflammatory cytokines, were produced by M1 macrophages. M2 macrophage produce the anti‑inflammatory cytokine IL-10 . Our results showed that proinflammatory factors were induced in the uterine cavity of obese patients associated with impaired autophagy.
5. Decidualization is impaired in patients with obesity
To investigate whether decidualization is impaired in the endometrium of obese patients, insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL) , two well-established markers of decidualization, mRNA expression levels at the late secretory phase of the menstrual cycle were detected. We found that these markers of decidualization were expressed at lower levels in endometrial tissue obtained from obese women compared with that from normal-weight women (Fig. 5A,C). Meanwhile, endometrial epithelial (ECC-1) cells were treated with ‘lean’ (2000 nmol/mol) or ‘obese’ (8000 nmol/mol) levels of lysine according to a previously established method of inducing decidualization . After decidualization, cells treated with ‘obese’ levels of lysine expressed significantly lower mRNA levels of IGFBP1 and PRL (Fig. 5B and D) than control cells. From these data, we concluded that exposure to high levels of lysine and obesity could impair decidualization.
6. Obesity disrupts endometrial stromal cell autophagic flux
Next, we assayed the autophagic flux of decidualization of ‘lean’ - vs. ‘obese’ - treated ECC-1 cells. BafA1 can be used to block autophagy to lysosomal degradation; therefore, an accumulation of LC3B-II in these conditions indicates an increase in autophagic flux. We found that levels of LC3B-II tended to increase while p62 (also known as sequestosome 1) decreased in lean decidualized cells compared with non-decidualized cells (Fig.6A). However, we observed a significantly lower level of LC3B-II and a higher level of p62 in obese-treated non-decidualized and decidualized ECC-1 cells compare with that in lean-treated ECC-1 cells (Fig. 6B). The amount of LC3B-II significantly increased upon BafA1 treatment in lean-treated decidual ECC-1 cells, suggesting that lysosomal degradation of LC3B-II was inhibited. However, this trend was not obvious in the obese-treated decidual ECC-1 cells (Fig. 6B). Western blotting analysis comparing autophagic flux in obese- vs. lean-treated cells from matched patients revealed impaired autophagic flux in obese patients compared with that in cells from lean women.