Depletion of oocyte dynamin-related protein 1 shows maternal-effect abnormalities in embryonic development

Abstract

Eggs contain about 200,000 mitochondria that generate adenosine triphosphate and metabolites essential for oocyte development. Mitochondria also integrate metabolism and transcription via metabolites that regulate epigenetic modifiers, but there is no direct evidence linking oocyte mitochondrial function to the maternal epigenome and subsequent embryo development. Here, we have disrupted oocyte mitochondrial function via deletion of the mitochondrial fission factor Drp1. Fission-deficient oocytes exhibit a high frequency of failure in peri- and postimplantation development. This is associated with altered mitochondrial function, changes in the oocyte transcriptome and proteome, altered subcortical maternal complex, and a decrease in oocyte DNA methylation and H3K27me3. Transplanting pronuclei of fertilized Drp1 knockout oocytes to normal ooplasm fails to rescue embryonic lethality. We conclude that mitochondrial function plays a role in establishing the maternal epigenome, with serious consequences for embryo development.

Fig. 1.. Oocyte-specific deletion of Drp1 and analysis of oocyte mitochondria.

(A to D) Oocyte-specific deletion of Drp1. DRP1 (green) and DNA (pink) in Drp1^fl/fl oocytes (A) but Drp1^Δ/Δ oocytes lacked DRP1 (B). Dashed circles represent oocyte margins. (C) DRP1 fluorescence quantification. AU, arbitrary unit. (D) Western blots of DRP1. Each lane contains 50 GV-stage oocytes from 4- to 5-week-old mice. β-Actin was used as a loading control. The experiments were repeated three times, and a representative image is shown. Mitochondrial morphology in GV-stage Drp1^fl/fl (E and F) and Drp1^Δ/Δ (G and H) oocytes. Branch length mean (I) and network branches mean (J) between Drp1^fl/fl and Drp1^Δ/Δ oocytes. Oocytes were labeled with mitotracker orange. Number of oocytes analyzed are shown (C, I, and J). (K) Increased mitochondrial ROS (mtROS) levels in Drp1^Δ/Δ oocytes. Numbers represent total number of oocytes. (L) Reduced MMP in Drp1^Δ/Δ oocytes compared to Drp1^fl/fl oocytes. Carbonyl cyanide-4-phenylhydrazone (FCCP) was used to demonstrate assay specificity. Numbers represent total number of oocytes. (M) Similar mitochondrial mass between Drp1^fl/fl and Drp1^Δ/Δ oocytes. MitoTracker Green signals in (L) were analyzed to calculate mitochondrial mass. ns, not significant. (N) Comparable mtDNA copy number between Drp1^fl/fl and Drp1^Δ/Δ oocytes. Numbers represent total number of oocytes. (O) Similar ATP levels between Drp1^Δ/Δ and Drp1^fl/fl oocytes. Numbers represent experimental replicates. Drp1^Δ/Δ oocytes have increased levels of FAD⁺⁺ (P) and NAD(P)H (Q) compared to Drp1^fl/fl controls. Total oocyte numbers shown. Representative autofluorescent images are also shown. (R) Comparable PBE rates in vitro. Numbers represent experimental replicates. (S to U) MII oocytes showing spindle (green) and DNA (blue). Chromosome spreads were measured as exemplified in (T) and (U). Total oocyte numbers shown in (S). Results show means ± SD and P values determined by two-tailed Student’s t test (C and I to S). F.C., fold change.

Fig. 2.. Preimplantation development of Drp1^matΔ/+ embryos.

(A) Schematic representation of mating schemes and the genotype of resultant embryos. (B to D) Comparison of mitochondrial mass between blastomeres of 2-cell embryos. One-cell zygotes harvested from gonadotropin-stimulated females and mated with WT males were cultured for 24 hours and labelled with MitoTracker Orange. Numbers represent total number of zygotes. (E to G) After gonadotropin stimulations, 4- to 5-week-old Drp1^fl/fl and Drp1^cKO mice were mated with WT males to harvest Drp1^fl/+ and Drp1^matΔ/+ one-cell zygotes. One-cell zygotes were cultured in vitro for 96 hours. Number of replicates are shown. Bla, blastocyst. (H to J) Blastocysts were labelled with CDX2 and OCT-4 to count the trophectoderm (TE) and inner cell mass (ICM), respectively. Representative maximum projection images of Drp1^fl/+ (I) and Drp1^matΔ/+ (J) blastocysts are shown. Number of blastocysts analyzed in (H) are shown. (K to M) Absolute requirement of DRP1 for preimplantation embryo development. The rate of parthenogenetic activation of Drp1^Δ/Δ oocytes was significantly reduced compared to Drp1^fl/fl controls (K). Numbers of experimental repeats are shown. Bla, blastocyst. Representative images of Drp1^fl/fl control (L) and Drp1^Δ/Δ (M) parthenotes at the end of culture. (N to V) Recovery of DRP1 and mitochondrial morphology in fertilized Drp1^matΔ/+ embryos. DRP1 expression was largely recovered in fertilized Drp1^matΔ/+ embryos (N). Number of blastocysts analyzed are shown. Immunofluorescence of DRP1 (cyan) was evident in Drp1^fl/+ (O) and Drp1^matΔ/+ (P) blastocysts. Unfertilized Drp1^fl/fl control parthenote expressed DRP1 (Q) but Drp1^Δ/Δ parthenote blastocysts completely lacked DRP1 immunofluorescence (R). Reflecting the DRP1 expression profiles, TMRM labelling revealed normal singular mitochondria in control fertilized Drp1^fl/+ (S) and Drp1^matΔ/+ (T) blastocysts. Unfertilized Drp1^fl/fl control parthenote also contained dispersed mitochondria (U) but Drp1^Δ/Δ parthenogenetic blastocysts had mitochondria aggregated into large clumps (V). Results show mean ± SD and P values determined by two-tailed Student’s t test (E, H, K, N).

Fig. 3.. Postimplantation death of Drp1^matΔ/+ embryos derived from Drp1^Δ/Δ oocytes.

Five-week-old Drp1^cKO and control Drp1^fl/fl females were mated with WT ^(+/+) males and assessed fetal development between E10.5 to E18.5 of pregnancy. (A) Uteri of 94% of plugged control Drp1^fl/fl females showed embryos as compared to only 60% of plugged Drp1^cKO females contained any embryo. Number of mice analyzed are shown. (B) Control females had an average of eight implants per pregnancy, whereas Drp1^cKO females had only four implants per pregnancy. Number of mice analyzed are shown. (C) Within confirmed pregnancies, there was a higher percentage of embryo resorption in Drp1^cKO females (76%) compared to only 3% in Drp1^fl/fl females. Number of mice analyzed are shown. While the uteri of E13.5 Drp1^fl/fl females showed normal developing embryos (D), the embryos in uteri of Drp1^cKO females were either dead (E, arrows) or severely growth retarded (E, arrowheads). Results show means ± SD (B and C). P value determined by chi-square test (A), two-tailed Student’s t test (B and C).

Fig. 4.. Altered gene expression profile of Drp1^Δ/Δ oocytes.

(A) Volcano plot showing DEGs (down-regulated, blue; up-regulated, red; and not different, black) between Drp1^fl/fl and Drp1^Δ/Δ oocytes. Highly dysregulated genes are labeled. (B) Heatmap of differential gene expression in Drp1^fl/fl and Drp1^Δ/Δ oocytes. CPM, counts per million. (C) Heatmap showing differential expression of epigenetic regulators in Drp1^fl/fl and Drp1^Δ/Δ oocytes. Number of single GV-stage oocytes per genotype used for RNA-seq analysis are shown (A to C). (D) Volcano plot showing differentially abundant proteins (down-regulated, blue; up-regulated, red; and not different, black) between MII-stage Drp1^fl/fl and Drp1^Δ/Δ oocytes. Highly dysregulated proteins are labeled. (E) Hierarchically clustered heatmap of relative abundancies of proteins in Drp1^fl/fl and Drp1^Δ/Δ oocytes. Number of replicates per genotype used for proteomic analysis are shown (D and E).

Fig. 5.. Altered epigenetic marks in the nuclei of Drp1^Δ/Δ oocytes and Drp1^matΔ/+ 2-cell zygotes.

Immunofluorescence (IF) showing 5mC (A, magenta), H3K27me3 (B, green), and merged (C) in Drp1^fl/fl oocytes. IF showing 5mC (D), H3K27me3 (E), and merged (F) in Drp1^Δ/Δ oocytes. (G) Quantification of 5mC fluorescence. (H) Quantification of H3K27me3 fluorescence. IF showing H3K27ac (I, green), DNA (J, blue), and merged (K) in Drp1^fl/fl oocytes. IF showing H3K27ac (L, green), DNA (M, blue), and merged (N) in Drp1^Δ/Δ oocytes. (O) Quantification of H3K27ac fluorescence. IF showing 5mC (P, green), H3K27me3 (Q, magenta) and merged (R) in Drp1^fl/+ 2-cell zygotes. IF showing 5mC (S), H3K27me3 (T) and merged (U) in Drp1^matΔ/+ 2-cell zygotes. (V) Quantification of 5mC fluorescence. (W) Quantification of H3K27me3 fluorescence. Results show mean ± SD and P values determined by two-tailed Student’s t test (G, H, O, V and W). Number of oocytes/zygotes analyzed are shown (G, H, O, V, and W).

Fig. 6.. Development after embryo transfer.

(A to D) Embryonic lethality is due to defective Drp1^Δ/Δ oocytes. (A) Schematic representation of embryo transfer experiment. Superovulated 5-week-old Drp1^fl/fl and Drp1^CKO females were mated with WT (^+/+) males to generate WT Drp1^fl/+embryos and heterozygous Drp1^matΔ/+ zygotes, respectively. One-cell zygotes harvested from the oviducts of plugged donor females were cultured in vitro for 24 hours until they reached the two-cell stage. Two-cell stage embryos were transferred to the oviducts of different WT pseudo-pregnant recipients. (B) Recipients culled between E15.5 and E20.5 of pregnancy revealed that eight of nine recipients receiving Drp1^fl/+ embryos became pregnant compared to only four of eight recipients receiving Drp1Drp1^matΔ/+ embryos. (C) Per-pregnancy implantation rate of Drp1^matΔ/+ embryos was markedly reduced than Drp1^fl/+ embryos. (D) Within confirmed pregnancies, there was a higher percentage of embryo resorption in Drp1^cKO females compared to Drp1^fl/fl females. (E to H) Embryo development after pronuclear swapping and embryo transfer. (E) Schematic representation of reciprocal pronuclear swapping between one-cell zygotes, in vitro culture, and embryo transfer. (F) Comparison of pregnancy rates after implanting KO Cyt and KO Pn zygotes. (G) Implantation rates of KO Pn and KO Cyt embryos. Within positive pregnancies, there was a higher percentage of resorption of KO Pn embryos compared to KO Cyt embryos (H). P values determined by chi-square test (B and F) and two-tailed Student’s t test (C, D, G, and H). Results show means ± SD (C, D, G, and H). Number of mice analyzed are shown (B to D and F to H).