FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

In Vivo-Matured Oocyte Resists Post-Ovulatory Aging through the Hub Genes DDX18 and DNAJC7 in Pigs.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.