Autophagy, which has the literal meaning of self-eating, is a cellular catabolic process executed by arrays of conserved proteins in eukaryotes. Autophagy is dynamically ongoing at a basal level, presumably in all cells, and often carries out distinct functions depending on the cell type. Therefore, although a set of common genes and proteins is involved in this process, the outcome of autophagic activation or deficit requires scrutiny regarding how it affects cells in a specific pathophysiological context. The uterus is a complex organ that carries out multiple tasks under the influence of cyclic changes of ovarian steroid hormones. Several major populations of cells are present in the uterus, and the interactions among them drive complex physiological tasks. Mouse models with autophagic deficits in the uterus are very limited, but provide an initial glimpse at how autophagy plays a distinct role in different uterine tissues. Herein, we review recent research findings on the role of autophagy in the uterine mesenchyme in mouse models.

Transcription factors govern diverse aspects of cell growth and differentiation as major switches of gene expression. Etv5, a member of the E26 transformation-specific family of transcription factors, has many stories to share when it comes to reproduction. Etv5 deficient mice show complex infertility phenotypes both in males and females. In males, the infertility phenotype exhibited by Etv5 deficiency is sexually dimorphic, and it involves both somatic cells and germ cells. In Etv5-/- female mice, the problem is more complicated by hormonal involvement. This review synthesizes old and new information on this versatile transcription factor-from the inadvertent discovery of its role in the testes to its newly discovered role in maintaining spermatogonial stem cells.
Animals ; Female ; Gene Expression ; Germ Cells ; Humans ; Infertility ; Male ; Mice ; Phenotype ; Reproduction ; Stem Cells ; Testis ; Transcription Factors

Objective: Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. Methods: All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. Results: SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. Conclusion Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

OBJECTIVE: Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. METHODS: Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. RESULTS: The survival rate of vitrified-warmed Atg7(f/f);Zp3-Cre (Atg7(d/d)) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7(f/f)) oocytes. Fertilization and development in the Atg7(d/d) oocytes were significantly lower than the Atg7(f/f) oocytes, comparable to the Atg5d/d oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7(d/d) MII oocytes when compared to fresh Atg7(d/d) oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. CONCLUSION: We confirmed that the LC3-positive signal is nearly absent in Atg7(d/d) oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.
Animals ; Autophagy ; Fertilization ; Fertilization in Vitro* ; Fluorescent Antibody Technique ; Genes, vif ; Metaphase ; Mice* ; Mice, Transgenic ; Oocytes* ; Organelles ; Recycling ; Survival Rate ; Vitrification*

OBJECTIVE: Under estrogen deficiency, blastocysts cannot initiate implantation and enter dormancy. Dormant blastocysts live longer in utero than normal blastocysts, and autophagy has been suggested as a mechanism underlying the sustained survival of dormant blastocysts during delayed implantation. Autophagy is a cellular degradation pathway and a central component of the integrated stress response. Reactive oxygen species (ROS) are produced within cells during normal metabolism, but their levels increase dramatically under stressful conditions. We investigated whether heightened autophagy in dormant blastocysts is associated with the increased oxidative stress under the unfavorable condition of delayed implantation. METHODS: To visualize ROS production, day 8 (short-term dormancy) and day 20 (long-term dormancy) dormant blastocysts were loaded with 1-microM 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). To block autophagic activation, 3-methyladenine (3-MA) and wortmannin were used in vivo and in vitro, respectively. RESULTS: We observed that ROS production was not significantly affected by the status of dormancy; in other words, both dormant and activated blastocysts showed high levels of ROS. However, ROS production was higher in the dormant blastocysts of the long-term dormancy group than in those of the short-term group. The addition of wortmannin to dormant blastocysts in vitro and 3-MA injection in vivo significantly increased ROS production in the short-term dormant blastocysts. In the long-term dormant blastocysts, ROS levels were not significantly affected by the treatment of the autophagy inhibitor. CONCLUSION: During delayed implantation, heightened autophagy in dormant blastocysts may be operative as a potential mechanism to reduce oxidative stress. Further, ROS may be one of the potential causes of compromised developmental competence of long-term dormant blastocysts after implantation.
Animals ; Autophagy* ; Blastocyst* ; Estrogens ; Mental Competency ; Metabolism ; Mice* ; Oxidative Stress ; Reactive Oxygen Species*