Fig. 1: Gm364 is an oocyte-rich transmembrane protein essential for female fertility.

A, B Ovarian immunofluorescence and quantification showed that Gm364 is more abundant in oocytes than in granular cells (GCs), and oocyte Gm364 is more abundant in antral follicles than in primary or secondary follicles. C Western blot of oocytes and GCs retrieved from antral follicles showed that Gm364 is more abundant in oocytes than in GCs. D Immunofluorescence showed that Gm364 is enriched on the oocyte membrane. Different Z-slices from the cortex to the cytoplasm are shown. DNA in blue, Gm364 in green. E. Blots showed that Gm364 protein levels gradually decreased during oocyte meiosis. F, G Through Cas9 technique, 55 bp was deleted from exon 1 of Gm364, which resulted in a frameshift and complete loss of Gm364 protein. H. Immunofluorescence showed that Gm364 protein in oocytes almost completely disappeared after Gm364 knockout. I, J Fertility assay from 8 to 44 weeks showed that Gm364 knockout greatly reduced the number of cumulative pups per female (I) and the number of cumulative pups per month (J). K–M Gm364 knockout significantly decreased the numbers of total follicles (TF), primordial follicles (PMF), secondary follicles (SF), and antral follicles (AF). Primary follicles, PF. Primordial follicles are arrow-pointed. DNA in blue, VASA in red. N, O Immunofluorescence and quantification in oocytes showed that Gm364 knockout significantly increased γ-H2AX signal dots within oocytes. DNA in blue, γ-H2AX in green. P, Q Immunofluorescence and quantification in ovaries showed that Gm364 knockout significantly increased γ-H2AX signal dots within the antral follicles. A series of Z-slices were aligned to show the clear difference (P). The number of γ-H2AX signal dots for each slice was counted and aligned into curves (Q). β-Actin or GAPDH was used as a loading control. Scale bar for (A), (K), and (P), 100 μm; scale bar for (D), (H), and (N), 20 μm. *Indicates p < 0.05.