Atg14 loss in the FRT results in infertility.

(A) Representative immunofluorescent images of uteri from pregnant mice at the indicated days of pregnancy stained with an ATG14-specific antibody (green). LE: luminal epithelium, G: glands, S: stroma, Scale bar: 200 µm. (B) Relative transcript levels of Atg14 mRNA in uteri from pregnant mice at indicated days of pregnancy. mRNA levels are normalized to levels of 18S m-RNA. Data are presented as mean ±SEM. (C) Relative mRNA levels of Atg14 in 8-week-old virgin control and cKO mice uteri, ovary, and liver (n=5). mRNA levels are normalized to levels of 18S mRNA. Data are presented as mean ±SEM; ***P<0.001, P>0.05, ns=non-significant. (D) Representative immunofluorescent images of ATG14 expression in different uterine compartments in control and Atg14 cKO mice. (E) (left panel) Relative number of pups/female and (right panel) and relative number of total pups/months of Atg14 control and cKO mice sacrificed after the breeding trial. Data are presented as mean ±SEM; P>0.05, ns=non-significant.

Six-month breeding trial of Atg14 control and cKO females with wild-type males

Six-month breeding trial of Foxj1/Atg14 control and cKO females with wild-type males

Atg14 is critical for embryo implantation, uterine receptivity, and stromal cell decidualization.

(A) Gross images of 5.0 dpc uteri of control and Atg14 cKO mice injected with Chicago Sky Blue dye to visualize implantation sites (denoted by black arrows) (left panel). H&E-stained cross-sections (4X & 40X) of 5.0 dpc uteri of control and Atg14 cKO mice to visualize embryo implantation (Middle panel). Immunofluorescence analysis of uterine tissues from control and Atg14 cKO mice, stained with MUC1 and KRT8 (right panel). (B) Representative immunofluorescence images of uteri from control and Atg14 cKO mice stained for Ki-67 following Oil or E2 or E2+P4 treatment (n=5 mice/group); scale bar: 100 μm. (C) Representative images of uteri (left) and uterine histology after artificial decidualization (middle). Immunofluorescence analysis of PH3 in decidualized horn of Control and Atg14 cKO mice. (D) Relative transcript levels of Atg14, and decidualization markers (Bmp2 and Wnt4) after artificial decidualization. (n=4-6 per group). Data are presented as mean ±SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls. 18S was used as an internal control. (F) Morphology of human endometrial stromal cells transfected with control or Atg14 siRNA followed by treatment with decidualization media and (G) qPCR analysis of ATG14 and the indicated decidualization markers. Data are presented as mean ±SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls.

Atg14 is critical for embryo transport in oviduct.

(A) Representative images (upper panel) and percentage of embryos (lower panel) collected at 4 dpc from the uteri or the oviducts of control or Atg14 cKO mice. (B) Histological analysis using H&E staining of the ampullary and isthmic region of the oviduct from control and Atg14 cKO female mice at 4 dpc (n=3 mice/genotype). (C) Embryos retrieved from the oviduct and uterus of super-ovulated Atg14 control or cKO mice at 4 dpc. (D) Immunofluorescence analysis of KRT8 (green), and α-SMA (Red), in oviduct of 4 dpc control and Atg14 cKO mice. (E) TEM of oviducts at 4 dpc from control and Atg14 cKO.

Atg14 loss in oviduct cilia is dispensable for embryo transport.

(A) Immunofluorescence analysis of acetylated a-tubulin (green), and DAPI (blue) in oviduct of 4 dpc control and Atg14 cKO mice. (B) Immunohistochemical analysis of FOXJ1 and PAX8 at 4 dpc. (C & D) Relative number of pups/femalsoyale and relative number of total pups/months of control and Foxj1/Atg14 cKO mice sacrificed after the breeding trial. Data are presented as mean ± SEM; P>0.05, ns=non-significant. (E) Gross images of 5.0 dpc uteri of control and Atg14 cKO mice injected with Chicago Sky Blue dye to visualize implantation sites (denoted by black arrows).

Atg14 promotes embryo transport in oviduct by preventing pyroptosis activation.

Immunofluorescence analysis of GSDMD (red) + KRT8 (green), Caspase-1 (red) + KRT8 (green) in oviducts of adult control and Atg14 cKO mice. Tissues were counterstained with DAPI (blue) to visualize nuclei; scale bars, 100 µm. (B) Immunohistochemical analysis of GSDMD in adult oviduct tissues. (C) Western blotting to show protein levels of Caspase-1, and GSDMD in oviduct tissues. β-actin is used as a loading control. (D) Relative transcript levels of Tnf-α and Cxcr3 in oviduct tissues. Data are presented as mean ±SEM. *P<0.05; **P<0.01; ***P<0.001 compared with controls. 18S was used as an internal control. (E) Experimental strategy for pyroptosis activation in pregnant female mice. (F) Embryos flushed from vehicle or polyphyllin VI treated D-4 pregnant females. (G) Percentage of embryos recovered from oviducts or uteri. (H) Graphical illustration to show embryo transport and pyroptosis regulation in oviduct.