Fig 2: LRRC4 deficiency triggers imbalance in mitochondrial dynamics. A) Heatmap of DEGs in mitochondrial dynamics and mitophagy based on transcriptome of GCs from WT and Lrrc4−/− mice. B) Representative fluorescent image showing co‐expression of mitochondria (red) and DRP1 (green) in NC, LRRC4−/− and Rescue KGN cells. Scale bar, 10 µm; magnified scale bar, 2 µm. C) The PCC and MCC analysis on the co‐expression of mitochondria and DRP1 signals in NC, LRRC4−/− and Rescue KGN cells (n = 30 biological replicates). D) Representative fluorescent image showing co‐expression of mitochondria (red) and MFN2 (green) in NC, LRRC4−/− and Rescue KGN cells. Scale bar, 10 µm; magnified scale bar, 2 µm. E) The PCC and MCC analysis on the co‐expression of mitochondria and MFN2 signals in NC, LRRC4−/− and Rescue KGN cells (n = 30 biological replicates). F) Representative fluorescent image showing co‐expression of mitochondria (red) and LC3B (green) in NC, LRRC4−/− and Rescue KGN cells. Scale bar, 10 µm; magnified scale bar, 2 µm. G) The PCC and MCC analysis on the co‐expression of mitochondria and LC3B signals in NC, LRRC4−/− and Rescue KGN cells (n = 30 biological replicates). H) Representative fluorescent image showing co‐expression of mitochondria (red) and LysoTracker (green) in NC, LRRC4−/− and Rescue KGN cells. Scale bar, 10 µm; magnified scale bar, 2 µm. I) The PCC and MCC analysis on the co‐expression of mitochondria and LysoTracker signals in NC, LRRC4−/− and Rescue KGN cells (n = 30 biological replicates). J) Representative fluorescent image showing co‐expression of mitochondria (red) and p62 (green) in NC, LRRC4−/− and Rescue KGN cells. Scale bar, 10 µm; magnified scale bar, 2 µm. K) The PCC and MCC analysis on the co‐expression of mitochondria and p62 signals in NC, LRRC4−/− and Rescue KGN cells (n = 30 biological replicates). L) Representative fluorescent images of mitophagic activity by mt‐Keima assays in NC, LRRC4−/− and Rescue KGN cells (n = 6 biological replicates). Cells treated with FCCP are positive controls. Scale bar, 10 µm. M) Representative electron microscopy images of mitochondria in NC, LRRC4−/− and Rescue KGN cells. Red arrow, normal mitochondria; yellow arrow, endoplasmic reticulum; green arrow, mitophagosome; blue arrow, autolysosome; black arrow, lysosome. Scale bar, 2 µm; magnified scale bar, 500 nm. N) Number of mitophagosome and autolysosome in NC, LRRC4−/− and Rescue KGN cells per microscopic field (n = 6 biological replicates). ##p < 0.01 means NC versus LRRC4−/− . Results are calculated with one‐way ANOVA. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: The dynamic expression pattern of LRRC4 in the ovary. A and B) Expression of LRRC4 in fetal germ cells (FGCs) and gonadal somatic cells (A) and different development phases of FGCs (B) based on GSE86146. Student's t‐test and one‐way ANOVA. C) Expression of LRRC4 in human ovarian cells during folliculogenesis based on GSE107746. C1, Primary Follicle; C2, Secondary Follicle; C3, Antral Follicle; C4, Preovulatory Follicle. D and E) Feature plots showing subsets in mouse ovarian cells (D) and LRRC4 expression in these subsets (E) based on GSE86146. F) Representative fluorescent image showing co‐expression of LRRC4 (green), FOXL2 (violet), and DDX4 (red) in mouse ovaries. Scale bar, 200 µm. G) Representative fluorescent images of LRRC4 expression in mouse ovaries during folliculogenesis. PmF, Primordial Follicle; PF, Primary Follicle; SF, Secondary Follicle; AF, Antral Follicle; PreF, Preovulatory Follicle. Scale bar for PmF, PF, and SF, 20 µm; Scale bar for AF and PreF, 50 µm. H) LRRC4 expression in ovarian cells from young and perimenopausal women based on GSE202601. Student's t‐test. I) LRRC4 expression in ovarian cells from mice at 3‐month‐old and 9‐month‐old based on GSE232309. J) LRRC4 protein expression of mouse ovaries from different ages (n = 3 biological replicates). K) Representative fluorescent images of LRRC4 expression in mouse ovaries from different ages. Scale bar for 3 days, 50 µm; Scale bar for 3, 8, 12 weeks and 16 months, 200 µm. L and N) Correlation analysis of LRRC4 expression with biomarkers of ovarian reserve. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: LRRC4 deficiency disturbs follicular development leading to premature ovarian insufficiency. A) Cumulative number of offspring in WT and Lrrc4−/− mice (n = 5 mice per group). B) Average litter size of WT and Lrrc4−/− mice (n = 5 mice per group). C) Representative images of ovarian morphology from 8‐week‐old WT and Lrrc4−/− mice. D) Ovary index values of 8‐week‐old WT and Lrrc4−/− mice (n = 8 mice per group). E) Representative images of ovaries from WT and Lrrc4−/− mice at 3‐week‐old, 8‐week‐old and 16‐month‐old by HE staining. Black arrows: PmF, yellow arrows: PF, green arrows: SF, and red arrows: AF. Scale bar for 3 weeks, 100 µm; Scale bar for 8 weeks and 16 months, 200 µm. F and G) Numbers of follicles in different developmental stages per ovary in 3‐week‐old (F) and 8‐week‐old WT and Lrrc4−/− mice (n = 7 mice per group). H) Relative mRNA expression of genes triggering primary follicle activation in ovaries from 3‐week‐old WT and Lrrc4−/− mice (n = 3 biological replicates). (I and J) Representative fluorescent images of Foxo3a I) and p‐rpS6 J) expression in 3‐week‐old WT and Lrrc4−/− mice. Scale bar, 20 µm. K to M) Serum levels of FSH (K), AMH (L) and E2 N) in 8‐week‐old WT and Lrrc4−/− mice (n = 8 mice per group). (N) Representative Masson staining images of ovaries from 8‐week‐old WT and Lrrc4−/− mice. Scale bar, 50 µm. Results are calculated with the Student's t‐test. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: LRRC4 promotes YAP ubiquitination degradation. A) Representative fluorescent image showing co‐expression of LRRC4 (violet) and YAP (green) in KGN cells. Scale bar, 10 µm; magnified scale bar, 5 µm. B) Co‐IP assays showing the interaction of exogenous LRRC4 and YAP in KGN cells (n = 3 biological replicates). C and D) Co‐IP assays showing the interaction of exogenous LRRC4 with endogenous (C) and exogenous (D) YAP in KGN cells (3 independent experiments) in KGN cells (n = 3 biological replicates). E) Relative mRNA expression of YAP in NC, LRRC4−/− and LRRC4‐OE KGN cells (n = 3 biological replicates). LRRC4‐OE KGN cells refer to KGN cells transfected with FLAG‐LRRC4 expression vector. Results are calculated with one‐way ANOVA. F) Protein expression of YAP in NC and LRRC4−/− KGN cells (n = 3 biological replicates). G) Protein expression of YAP in KGN cells transfected with the increasing amount of FLAG‐LRRC4 expression vector (n = 3 biological replicates). H) Representative fluorescent images of YAP (red) in NC and LRRC4−/− KGN cells. Scale bar, 50 µm; magnified scale bar, 10 µm. I) Protein expression of YAP in LRRC4‐OE KGN cells treated with or without the proteasome inhibitor MG132 (20 µM, 6 h) (n = 3 biological replicates). J) Protein expression of YAP in NC and LRRC4−/− KGN cells treated with 30 µg mL−1 for the indicated times (n = 3 biological replicates). K) Protein expression of YAP in control and LRRC4‐OE KGN cells treated with 30 µg mL−1 CHX for the indicated times (n = 3 biological replicates). L) The ubiquitination of YAP in NC and LRRC4−/− KGN cells (n = 3 biological replicates). M) The ubiquitination of YAP in NC and LRRC4‐OE KGN cells (n = 3 biological replicates). N and O) K48‐linked (N) and K63‐linked (O) YAP polyubiquitination in NC and LRRC4‐OE KGN cells (n = 3 biological replicates). Data are presented as mean ± SEM. ns, not significant.