Fig 2: cRNA microinjection of Mad2l1bp accelerated or rescued the meiotic division in mouse germinal vesicle (GV) oocytes.(A) A schematic diagram depicting the time points of distinctive events through meiotic progression in mouse oocytes. cRNAs were microinjected into GV oocytes after 44 hr of PMSG priming. (B) Representative morphology of oocytes after microinjection with cRNAs encoding mock GFP, mouse Mad2l1bp WT, and Mad2l1bp Mut (equivalent to human p.R285*). Non-injected group was treated similarly to the other three groups without cRNA microinjection. Images were taken 14 hr following release from milrinone inhibitor. Scale bar, 100 µm. (C and D) Kinetic recordings showing the percentages of oocytes with GV breakdown (GVBD) (C), and the oocyte polar body extrusion (PBE) rate (D) through the time-lapse imaging experiment. The total numbers of oocytes microinjected were labeled as indicated. The experiments were performed in biological triplicates. Data are presented as mean ± SEM. (E) Representative morphology of oocytes after cRNA co-microinjection of Mad2 supplemented with WT or Mad2l1bp Mut (equivalent to human p.R285*) into the mouse GV oocytes. Images were taken 14 hr after release from milrinone. Scale bar, 100 µm. (F) Comparison of the percentages of oocytes with GVBD and PBE among the four microinjection groups in (E). The total numbers of oocytes microinjected were labeled as indicated. The experiments were performed in biological triplicates. The statistics were performed with Student’s t-test. Data are presented as mean ± SEM. ‘***‘ indicates p<0.001. Figure 3—source data 1.cRNA microinjection of full-length or truncated Mad2l1bp uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Figure 3—source data 2.Co-injection of Mad2 cRNAs with Mad2l1bp cRNAs (WT vs p.R285* mutant) indicated the Mad2l1bp variant lost its function in vivo as compared with its WT counterpart.

Fig 3: Identification of pathogenic variants in MAD2L1BP from three unrelated families.(A) Pedigrees of three female patients with oocyte maturation arrest at the MI stage from three unrelated families. The first case was from a consanguineous family, while the other two were not. The patients carried homozygous c.853C>T [p.R285*] and c.541C>T [p.R181*] variants from Family 1 and Family 3, respectively, while the patient from Family 2 had a compound heterozygous mutation c.518delT and c.21–94G>A as indicated. The chromatograms below show the Sanger sequencing results of the PCR-amplified fragments containing the respective variants in each family. (B) Phylogenetic conservation of the identified amino acid mutations in MAD2L1BP. The positions of all mutations are indicated in the schematic genomic structure of MAD2L1BP. The MAD2-interacting site and the seatbelt self-interaction region for MAD2L1BP are labeled in the secondary structure of MAD2L1BP protein at the bottom. All identified mutations residing in Exon 4 of MAD2L1BP generated the premature STOP codon, likely resulting in truncated proteins. (C) The representative morphology of normal and affected individual oocytes. The normal MI and MII oocytes are shown in the top panel. The red arrow points to the first polar body (PB1). Oocytes retrieved from Family 1 (II-1) and Family 2 (II-1) were arrested at the MI stage. The asterisks indicate the oocytes with a magnified view in the right panel. MI: metaphase I; MII: metaphase II. Scale bar, 80 µm.

Fig 4: Adverse impacts of MAD2L1BP variants on mRNA expression and protein function.(A–B) Quantitative PCR (qPCR) assays comparing the mRNA expression levels of MAD2L1BP in peripheral blood samples from Family 1 and Family 2 as indicated. The experiments were performed in technical triplicates. (C) qPCR assay showing the mRNA expression levels of MAD2 in individual blood samples from Family 1. The experiments were performed in technical triplicates. (D) The ectopic protein expression levels of Myc-MAD2, Flag-MAD2L1BP WT(wild-type), and Flag-MAD2L1BP MUT (p.R285*) by immunoblotting following transfection of individual plasmids into 293T cells in culture. Full-length and truncated MAD2L1BP protein can be seen in the panel. (E) Co-immunoprecipitation (Co-IP) assays demonstrating that the truncated MAD2L1BP MUT (p.R285*) lost interaction with MAD2, as compared with MAD2L1BP WT, when co-transfected into 293T cells in vitro. (F) Ribbon and topological diagrams showing the structure of MAD2L1BP WT (PDB ID: 2QYF) and the predicted MAD2L1BP MUT (p.R285*). Notably, MAD2L1BP MUT (p.R285*) lacks a C-terminal seatbelt configuration (highlighted in red) resulting in structural instability. Figure 2—source data 1.Blots for detecting Myc-MAD2, Flag-MAD2L1BP WT, Flag-MAD2L1BP MUT protein expression. Figure 2—source data 2.Co-immunoprecipitation (Co-IP) blots for detecting interaction between MAD2 and MAD2L1BP WT or MAD2L1BP MUT. Figure 2—source data 3.The original file of blots for Figure 2D. Figure 2—source data 4.The original file of blots for Figure 2E.