Atg14 loss in the FRT results in infertility.

(A) Representative immunofluorescent images of uteri from pregnant mice (n=3) at the indicated days of pregnancy stained with an ATG14-specific antibody (green). LE: luminal epithelium, G: glands, S: stroma, Scale bar: 100 µm. Rabbit IgG was used as an isotype control for staining. (B) Relative transcript levels of Atg14 mRNA in uteri from pregnant mice (n=3-5) at indicated days of pregnancy. mRNA levels are normalized to levels of 18S m-RNA. Data are presented as mean ±SEM; **P<0.01, P>0.05, ns=non-significant. (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 (n=5) and Atg14 cKO mice (n=5). LE: luminal epithelium, G: glands, S: stroma. (E) (left panel) Relative number of pups/female/litter and (right panel) and relative number of total pups/months of Atg14 control (n=4) and cKO mice (n=5) sacrificed after the breeding trial. Data are presented as mean ±SEM; ***P<0.001; P>0.05, ns=non-significant.

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

Atg14 is critical for embryo implantation, and uterine receptivity.

(A) Gross images of 5.0 dpc uteri of control (n=5) and Atg14 cKO mice (n=5) 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 (n=5) and Atg14 cKO (n=5) mice to visualize embryo implantation (Middle panel). The asterisk denotes the embryo. Immunofluorescence analysis of uterine tissues from control (n=5) and Atg14 cKO mice (n=5), stained with MUC1 and KRT8 (right panel). LE: luminal epithelium, G: glands, S: stroma. (B) Representative immunofluorescence images of uteri from control (n=5) and Atg14 cKO mice (n=5) stained for Ki-67 following Oil or E2 or E2+P4 treatment (n=5 mice/group); scale bar: 100 μm. LE: luminal epithelium, G: glands, S: stroma. (C) Immunofluorescence analysis of KRT8 (green), MUC1 (Red), and (D) Ki-67 (red) in the uteri of 4 dpc control and Atg14 cKO mice; scale bars, 100 µm. (E) Relative transcript levels of Lif, Mcm2, Ccnd1, and Fgf18 in control and cKO uteri at 4dpc. mRNA levels are normalized to levels of 18S m-RNA. Data are presented as mean ±SEM; ns=non-significant. (F) Levels of steroid hormones estradiol and progesterone from serum collected during euthanasia of 4 dpc control or cKO mice.

Atg14 is critical for embryo transport in oviduct.

(A) Representative images (upper panel) and (B) Percentage of embryos (lower panel) collected at 4 dpc from the uteri or the oviducts of control (n=6) or Atg14 cKO mice (n=6-8). (C) Percentage of blastocysts, morulae, developmentally delayed or nonviable embryos collected from Atg14 control mice uteri and Atg14 cKO mice oviducts at 4 dpc. (D) 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). (E) Embryos retrieved from the oviduct and uterus of super-ovulated Atg14 control or cKO mice at 4 dpc (n=3 mice/genotype). (F) Immunofluorescence analysis of KRT8 (green), and α-SMA (Red), in the oviduct of 4 dpc control (n=5) and Atg14 cKO mice (n=5) ampulla (upper panel) and isthmus (lower panel).

Atg14 loss in oviduct cilia is dispensable for embryo transport.

(A) Immunofluorescence analysis of acetylated α-tubulin (green), and DAPI (blue) in oviduct of 4 dpc control (n=5) and Atg14 cKO mice (n=5). (B) Immunohistochemical analysis of FOXJ1 and PAX8 at 4 dpc (n=5). (C) Immunohistochemical analysis of acetylated α-tubulin in 8-week-old control (n=5) and Foxj1/Atg14 cKO mice (n=5). Images are taken at 6X and 40X. Scale bar: 100µm and 10 µm. (D & E) Relative number of pups/females/litter and relative number of total pups/months of control (n=5) and Foxj1/Atg14 cKO mice (n=5) sacrificed after the breeding trial. Data are presented as mean ± SEM; P>0.05, ns=non-significant. (F) Gross images of 5.0 dpc uteri of control (n=3) and Foxj1/Atg14 cKO mice (n=3) injected with Chicago Sky Blue dye to visualize implantation sites (denoted by black arrows).

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

>ATG14 facilitates embryo transport in the oviduct by preserving mitochondrial integrity and inhibiting the activation of pyroptosis.

(A) Transmission electron microscopy of oviducts at 4 dpc from control (n=3) and Atg14 cKO (n=3). Black arrowheads in control oviducts show small, compact mitochondria with tight cristae. Red arrowheads in cKO oviducts show abnormally enlarged mitochondria with loose cristae. (B) Immunofluorescence analysis of TOM20 (green) and Cytochrome C (red) in Atg14 control and cKO oviducts cryo-sections. The inset shows the zoomed image of the selected area from control and cKO oviducts, Arrowheads in cKO inset oviduct section show the leaked cytochrome C in the cytoplasm. Tissues were counterstained with DAPI (blue) to visualize nuclei; scale bars, 10 µm. (C) Relative transcript levels of Opa1, Cox4i2, Drp1, and Pink1 in oviduct tissues. Data are presented as mean ±SEM. *P<0.05; **P<0.01 compared with controls. 18S was used as an internal control (D) Immunofluorescence analysis of GSDMD (red) + KRT8 (green), Caspase-1 (red) + KRT8 (green) in oviducts of adult control (n=5), and Atg14 cKO mice (n=5). Tissues were counterstained with DAPI (blue) to visualize nuclei; scale bars, 100 µm. (E) Immunohistochemical analysis of GSDMD expression in adult oviduct tissues (left panel). The middle and -right panels show the zoom-in images to show the relative GSDMD expression in the ampulla and isthmus section from control and cKO oviducts. (F) Immunohistochemical analysis of GSDMD expression in adult uterus and ovary tissues (G) Western blotting to show protein levels of Caspase-1, and GSDMD in oviduct tissues. β-actin is used as a loading control. (H) 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.

Pharmacological activation of pyroptosis in the oviduct inhibits embryo transport.

(A) Experimental strategy for pyroptosis activation in pregnant female mice (B) Embryos flushed from the vehicle (n=3) or polyphyllin VI treated (n=3) D-4 pregnant females. (C) Percentage of embryos recovered from oviducts or uteri. (D) Percentage of blastocysts, morulae, developmentally delayed or nonviable embryos collected from vehicle or Polyphyllin IV oviducts at 4 dpc. (E) Immunohistochemistry to show GSDMD expression in the isthmus section of polyphyllin IV-treated and vehicle-treated mice. 40X objective, Scale bar: 5 µm. (F) Graphical illustration to show embryo transport and pyroptosis regulation in the oviduct.