MATR3 in growing oocyte regulates female reproduction in mice.

A Immunofluorescence showing MATR3 levels in mouse oocytes. B Western blot results showing MATR3 expression in mouse oocytes. Total proteins from 200 oocytes were loaded in each lane. GAPDH served as a loading control. C Relative expression level of MATR3 to β-Actin. D Immunofluorescence showing MATR3 levels in human oocytes. E Representative image of follicles and oocytes from NC and si-Matr3. F Immunohistochemistry results showing MATR3 expression in oocytes. Arrows indicate oocytes in primordial follicles. G Cumulative number of pups from Ctrl (n = 6) and cKO (n = 3) female. H Percentage of antral follicles in e. n ≥ 33 per group. I MII ratio of oocytes collected from e. n ≥ 33 per group. Scale bars: 20 μm in A, B and C, 40 μm in E and F. Data are represented as mean±SD. ***P < 0.001, **P < 0.01, *P < 0.05, n.s., not significant.

MATR3 was required to support oocyte growth.

A Hematoxylin staining of PD23 ovaries after gonadotropins injection. B Number of follicles on PD23 ovaries after gonadotropins injection. n = 3. C Morphology and number of oocytes collected from oviducts of PD23 mice after superovulation. n = 3. D Morphology of oocytes derived from PD23 mice primed with PMSG for 48 h. n = 3. E and F Diameter of oocytes derived from PD23 mice primed with PMSG for 48 h. n = 3. G NSN and SN ratio of oocytes derived from PD23 mice primed with PMSG for 48 h. n = 3. H MII ratio of oocytes derived from PD23 mice primed with PMSG for 48 h. n = 3. I Hematoxylin staining of PD7, PD14, PD23, PD35 ovaries. n =3. J Number of total follicles in PD7, PD14, PD23, PD35 ovaries. n = 3. K Cell proliferation indicated by Ki67-positive GCs in mice ovaries. n = 3. L Statistics of AF/SF on PD23 and PD35 ovaries. n = 3. M Number of SFs in PD7, PD14, PD23, PD35 ovaries. n = 3. Scale bars: 40 μm in C and E, 120 μm in A, I and K. Data are represented as mean±SD. ***P < 0.001, **P < 0.01, *P < 0.05, n.s., not significant.

Matr3 knockdown results in the reduction of transcriptional activity.

A Volcano plot shows DEGs (upregulated, red; downregulated, blue) in si-Matr3 oocytes compared to the NC. B Key GO enrichment of all DEGs in GOs. C TOP10 terms of downregulated DEGs in GOs. D EU staining (green) in GO collected from NC and si-Matr3. n ≥20. E Quantification of the mean fluorescence intensity of EU in oocytes. F RT-qPCR results showing Tbpl2 mRNA level in GOs. G Western blotting results showing RNAPII protein level in GOs. H H3K9me1 (red), H3K9me3 (red), H3K27me3 (red), H3K4me3 (green), staining in GO collected from NC and si-Matr3. n ≥20. I H3K9me2 staining (red) in GO collected from NC and si-Matr3. n ≥ 37. J Quantification of the mean fluorescence intensity of H3K9me2 in oocytes. Scale bar: 20 μm. Data are represented as mean±S.D. ***P < 0.001, **P < 0.01.

MATR3 controls oocyte growth by regulating GDF9 level.

A RT-qPCR results showing Gdf9 mRNA level in PD14 ovaries. B and C Western blotting results showing GDF9, SMAD3 and p-SMAD3 protein levels in PD14 ovaries. D p-SMAD3 staining (green) in PD14 ovaries. E GDF9 staining (red) in GOs from NC and si-Matr3. n ≥68. F Quantification of the mean fluorescence intensity of GDF9 in oocytes. G Representative image of follicles and oocytes from NC, si-Matr3, si-Matr3+GDF9, GDF9. H Percentage of antral follicles and MII oocytes in G. n≥3 per group. Scale bar: 40 μm in D and E, 80 μm in G. Data are represented as mean±S.D. ***P < 0.001, **P < 0.01, *P < 0.05, n.s., not significant.

MATR3 promoted GDF9 by recruiting KDM3B.

A KDM3B staining (red) in GO collected from NC and si-Matr3. n ≥18. B Quantification of the mean fluorescence intensity of KDM3B (A) in oocytes. C and D CO-IP showing protein interactions between MATR3 and KDM3B in HEK293T cells. E CO-IP results after HEK293T cells were treated with pCDNA3.1-EGFP, pCDNA3.1-MATR3-EGFP, pCDNA3.1- ΔRRM2-EGFP or pCDNA3.1- Δ pNLS4-EGFP plasmid. F and G Live-cell imaging of oocytes after injecting Matr3-egfp mRNA for 12 h. H MATR3 (red) and GDF9 (green) staining in GO which had knocked down MATR3 protein before injected Matr3-egfp, Δrrm2-egfp, Δpnls4-egfp. n ≥36. I Quantification of the mean fluorescence intensity of GDF9 (H) in oocytes. Scale bar: 40 μm. Data are represented as mean±SD. ***P < 0.001, n.s., not significant.

MATR3 is required for the OO-Mvi in mouse oocyte by regulating Rdx mRNA level.

A Pie chart showing the proportion of the reads values of 10-12-day-mice MATR3 LACE-seq. B Table showing the base sequences to which MATR3 mainly binds. C and E Venn diagrams (c) and table (E) showing overlapping genes binding with MATR3, upregulated genes and downregulated genes in scRNA-seq. D Key GO enrichment of all DEGs in LACE-seq. F IGV snapshot of MATR3 peaks distribution in 10-12-day-mice ovaries. G and H RIP showing the interaction between MATR3 and Rdx mRNA in 10-12-day-mice ovaries. Western blotting showing the reliability of MATR3 antibody (G). Enrichment degrees of MATR3 on the Rdx mRNA respectively (H). IgG served as the negative control and Input (2%) served as the positive control. n = 3. I RT-qPCR results showing Rdx mRNA level in PD14 oocytes. J p-ERM staining (green) showing the OO-Mvi in SFs and AFs from Ctrl and cKO. Scale bar: 40 μm. Data are represented as mean±SD. ***P < 0.001.

A proposed model: MATR3 regulates the synthesis and secretion of OSFs, like GDF9, thereby ensuring GO growth.

Under physiological conditions, MATR3 in the growing oocytes promotes the transcription of a large number of genes required for cell growth. When promoting the transcription of the Gdf9 gene, MATR3 localizes in the nucleus through the pNLS4 domain to recruit KDM3B, thereby promoting the high-level expression of Gdf9. In ensuring the secretion of GDF9, MATR3 directly binds to Rdx mRNA to promote the growth of microvilli, which in turn lays the foundation for ensuring the secretion of factors such as GDF9 and establishing the physical connection between oocytes and somatic cells.