Search

Formin-like 3 (FMNL3) is a member of the formin-likes (FMNLs), which belong to the formin family. As an F-actin nucleator, FMNL3 is essential for several cellular functions, such as polarity control, invasion, and migration. However, the roles of FMNL3 during oocytes meiosis remain unclear. In this study, we investigated the functions of FMNL3 during mouse oocyte maturation. Our results showed that FMNL3 mainly concentrated in the oocyte cortex and spindle periphery. Depleting FMNL3 led to the failure of polar body extrusion, and we also found large polar bodies in the FMNL3-deleted oocytes, indicating the occurrence of symmetric meiotic division. There was no effect of FMNL3 on spindle organization; however, we observed spindle migration defects at late metaphase I, which might be due to the decreased cytoplasmic actin. Microinjecting Fmnl3-EGFP mRNA into Fmnl3-depleted oocytes significantly rescued these defects. In addition, the results of co-immunoprecipitation and the perturbation of protein expression experiments suggested that FMNL3 interacted with the actin-binding protein FASCIN for the regulation of actin filaments in oocytes. Thus, our results provide the evidence that FMNL3 regulates FASCIN for actin-mediated spindle migration and cytokinesis during mouse oocyte meiosis.

Mammalian oocyte maturation is a unique asymmetric division, which is mainly because of actin-based spindle migration to the cortex. In the present study, we report that a kinesin motor KIFC1, which is associated with microtubules for the maintenance of spindle poles in mitosis, is also involved in actin dynamics in murine oocyte meiosis, co-localizing with microtubules during mouse oocyte maturation. Depletion of KIFC1 caused the failure of polar body extrusion, and we found that meiotic spindle formation and chromosome alignment were disrupted. This might be because of the effects of KIFC1 on HDAC6 and NAT10-based tubulin acetylation, which further affected microtubule stability. Mass spectroscopy analysis revealed that KIFC1 also associated with several actin nucleation factors and we found that KIFC1 was essential for the distribution of actin filaments, which further affected spindle migration. Depletion of KIFC1 leaded to aberrant expression of formin 2 and the ARP2/3 complex, and endoplasmic reticulum distribution was also disturbed. Exogenous KIFC1 mRNA supplement could rescue these defects. Taken together, as well as its roles in tubulin acetylation, our study reported a previously undescribed role of kinesin KIFC1 on the regulation of actin dynamics for spindle migration in mouse oocytes.

Leucine-rich-repeat kinase 2 (LRRK2) belongs to the Roco GTPase family and is a large multidomain protein harboring both GTPase and kinase activities. LRRK2 plays indispensable roles in many processes, such as autophagy and vesicle trafficking in mitosis. In this study, we showed the critical roles of LRRK2 in mammalian oocyte meiosis. LRRK2 is mainly accumulated at the meiotic spindle periphery during oocyte maturation. Depleting LRRK2 led to the polar body extrusion defects and also induced large polar bodies in mouse oocytes. Mass spectrometry analysis and co-immunoprecipitation results showed that LRRK2 was associated with several actin-regulating factors, such as Fascin and Rho-kinase (ROCK), and depletion of LRRK2 affected the expression of ROCK, phosphorylated cofilin, and Fascin. Further analysis showed that LRRK2 depletion did not affect spindle organization but caused the failure of spindle migration, which was largely due to the decrease of cytoplasmic actin filaments. Moreover, LRRK2 showed a similar localization pattern to mitochondria, and LRRK2 was associated with several mitochondria-related proteins. Indeed, mitochondrial distribution and function were both disrupted in LRRK2-depleted oocytes. In summary, our results indicated the critical roles of LRRK2 in actin assembly for spindle migration and mitochondrial function in mouse oocyte meiosis.

Mammalian oocyte maturation involves a unique asymmetric cell division, in which meiotic spindle formation and actin filament-mediated spindle migration to the oocyte cortex are key processes. Here, we report that the vesicle trafficking regulator, RAB35 GTPase, is involved in regulating cytoskeleton dynamics in mouse oocytes. RAB35 GTPase mainly accumulated at the meiotic spindle periphery and cortex during oocyte meiosis. Depletion of RAB35 by morpholino microinjection led to aberrant polar body extrusion and asymmetric division defects in almost half the treated oocytes. We also found that RAB35 affected SIRT2 and αTAT for tubulin acetylation, which further modulated microtubule stability and meiotic spindle formation. Additionally, we found that RAB35 associated with RHOA in oocytes and modulated the ROCK–cofilin pathway for actin assembly, which further facilitated spindle migration for oocyte asymmetric division. Importantly, microinjection of Myc-Rab35 cRNA into RAB35-depleted oocytes could significantly rescue these defects. In summary, our results suggest that RAB35 GTPase has multiple roles in spindle stability and actin-mediated spindle migration in mouse oocyte meiosis.

Objectives RAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP‐bound inactive state and a GTP‐bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation. Materials and methods Microinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real‐time RT‐PCR were utilized for the study. Results Our results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection. Conclusions Taken together, our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation., RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 caused large polar bodies, indicating the failure of asymmetric division. Absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection. Our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.

Mammalian oocyte quality is critical for fertilization and early embryo development. The type B trichothecene nivalenol (NIV) is a mycotoxin produced by Fusarium oxysporum, and it is commonly found with deoxynivalenol in contaminated food or feed. NIV has been shown to affect the immune system and female reproductive system, cause emesis and growth retardation. Here, we investigated the toxicity of NIV on mouse oocyte quality, as well as the protective effects of melatonin on the NIV-exposed oocytes. We found NIV exposure caused meiotic arrest and further induced the failure of polar body extrusion in mouse oocytes. Transcriptome analysis data showed that NIV exposure altered the expression of multiple pathway-related genes in oocytes, indicating its wide toxicity on oocyte maturation. Based on the RNA-seq data, we showed that NIV exposure induced oxidative stress and caused DNA damage in oocytes. Besides, autophagy, and early apoptosis were also found in NIV-exposed oocytes. Treatment with melatonin significantly ameliorated these defects through its effects on ROS level. Thus, our results demonstrated that exposure to NIV affected oocyte quality and melatonin treatment could reduce the defects caused by NIV in mouse oocytes.

Infertility in humans at their reproductive age is a world-wide problem. Oocyte in vitro maturation (IVM) is generally used in such cases to acquire the embryo in assisted reproductive technology (ART). However, the differences between an in vivo (IVO) and IVM culture environment in the RNA expression profile in oocytes, remains unclear. In this study, we compared the global RNA transcription pattern of oocytes from in vitro and in vivo maturation. Our results showed that 1,864 genes differentially expressed between the IVO and IVM oocytes. Among these, 1,638 genes were up-regulated, and 226 genes were down-regulated, and these changes were mainly divided into environmental adaption, metabolism, and genetic expression. Our detailed analysis showed that the expression of genes that belonged to metabolism-related processes such as energy metabolism, nucleotide metabolism, and carbohydrate metabolism was changed; and these genes also belonged to organismal systems including environmental adaptation and the circulatory system; moreover, we also found that the relative gene expression of genetic expression processes, such as protein synthesis, modification, and DNA replication and repair were also altered. In conclusion, our data suggests that in vitro maturation of mouse oocyte resulted in metabolism and genetic expression changes due to environmental changes compared with in vivo matured oocytes.

GBF1 [Golgi brefeldin A (BFA) resistance factor 1] is a member of the guanine nucleotide exchange factors Arf family. GBF1 localizes at the cis-Golgi and endoplasmic reticulum (ER)-Golgi intermediate compartment where it participates in ER-Golgi traffic by assisting in the recruitment of the coat protein COPI. However, the roles of GBF1 in oocyte meiotic maturation are still unknown. In the present study, we investigated the regulatory functions of GBF1 in mouse oocyte organelle dynamics. In our results, GBF1 was stably expressed during oocyte maturation, and GBF1 localized at the spindle periphery during metaphase I. Inhibiting GBF1 activity led to aberrant accumulation of the Golgi apparatus around the spindle. This may be due to the effects of GBF1 on the localization of GM130, as GBF1 co-localized with GM130 and inhibiting GBF1 induced condensation of GM130. Moreover, the loss of GBF1 activity affected the ER distribution and induced ER stress, as shown by increased GRP78 expression. Mitochondrial localization and functions were affected, as the mitochondrial membrane potential was altered. Taken together, these results suggest that GBF1 has wide-ranging effects on the distribution and functions of Golgi apparatus, ER, and mitochondria as well as normal polar body formation in mouse oocytes.

Protein regulator of cytokinesis 1 (PRC1) is a microtubule bundling protein that is involved in the regulation of the central spindle bundle and spindle orientation during mitosis. However, the functions of PRC1 during meiosis have rarely been studied. In this study, we explored the roles of PRC1 during meiosis using an oocyte model. Our results found that PRC1 was expressed at all stages of mouse oocyte meiosis, and PRC1 accumulated in the midzone/midbody during anaphase/telophase I. Moreover, depleting PRC1 caused defects in polar body extrusion during mouse oocyte maturation. Further analysis found that PRC1 knockdown did not affect meiotic spindle formation or chromosome segregation; however, deleting PRC1 prevented formation of the midzone and midbody at the anaphase/telophase stage of meiosis I, which caused cytokinesis defects and further induced the formation of two spindles in the oocytes. PRC1 knockdown increased the level of tubulin acetylation, indicating that microtubule stability was affected. Furthermore, KIF4A and PRC1 showed similar localization in the midzone/midbody of oocytes at anaphase/telophase I, while the depletion of KIF4A affected the expression and localization of PRC1. The PRC1 mRNA injection rescued the defects caused by PRC1 knockdown in oocytes. In summary, our results suggest that PRC1 is critical for midzone/midbody formation and cytokinesis under regulation of KIF4A in mouse oocytes.

Background Environmental pollution induces oxidative stress and apoptosis in mammalian oocytes, which can cause defects in reproduction; however, the molecular regulation of oxidative stress in oocytes is still largely unknown. In the present study, we identified that dynamin-related protein 1 (DRP1) is an important molecule regulating oocyte mitochondrial function and preventing oxidative stress/apoptosis. DRP1 is a member of the dynamin GTPase superfamily localized at the mitochondrial-endoplasmic reticulum interaction site, where it regulates the fission of mitochondria and other related cellular processes. Results Our results show that DRP1 was stably expressed during different stages of porcine oocyte meiosis, and might have a potential relationship with mitochondria as it exhibited similar localization. Loss of DRP1 activity caused failed porcine oocyte maturation and cumulus cell expansion, as well as defects in polar body extrusion. Further analysis indicated that a DRP1 deficiency caused mitochondrial dysfunction and induced oxidative stress, which was confirmed by increased reactive oxygen species levels. Moreover, the incidence of early apoptosis increased as detected by positive Annexin-V signaling. Conclusions Taken together, our results indicate that DRP1 is essential for porcine oocyte maturation and that a DRP1 deficiency could induce mitochondrial dysfunction, oxidative stress, and apoptosis.

Nonylphenol (NP) is a chemical raw material and intermediate which is mainly used in the production of surfactants, lubricating oil additives and pesticide emulsifiers. NP is reported to be toxic on the immune system, nervous system and reproductive system due to its binding to estrogen receptors. However, the toxicity of NP on mammalian oocyte quality remains unclear. In present study, we explored the effects of NP exposure on mouse oocyte maturation. Our results showed that 4 weeks of NP exposure increased the number of atresia follicles and decreased oocyte developmental competence. Transcriptomic analysis indicated that NP exposure altered the expression of more than 800 genes in oocytes, including multiple biological pathways. Subcellular structure examination indicated that NP exposure disrupted meiotic spindle organization and caused chromosome misalignment. Moreover, aberrant mitochondrial distribution and decreased membrane potential were also observed, indicating that NP exposure caused mitochondria dysfunction. Further analysis showed that NP exposure resulted in the accumulation of reactive oxygen species (ROS), which causes oxidative stress; and the NP-exposed oocytes showed positive Annexin-V signal, indicating the occurrence of early apoptosis. In summary, our results indicated that NP exposure reduced oocyte quality by affecting cytoskeletal dynamics and mitochondrial function, which further induced oxidative stress and apoptosis in mice.

Citrinin (CTN) is a secondary fungal metabolite produced by several species of Aspergillins and Penicillins, and it is widely found in vegetable-derived foods such as cereals and fermented rice-based food supplements. Previous studies indicated that CTN had immunotoxicity, hepatotoxicity, nephrotoxicity, and reproductive toxicity, which caused severe effects on human and animal health. However, the potential toxicity of CTN on the organelles of mouse oocytes is still unclear. In this study, we showed that the exposure to 30 μM CTN significantly reduced the developmental capacity of mouse oocytes. Our results revealed that mitochondria exhibited abnormal distribution and mitochondrial membrane potential decreased under CTN exposure. And the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, which is accompanied by the occurrence of ER stress, showing with increased GRP78 expression. We also found that similar with ER, the Golgi apparatus showed homogenous localization pattern after CTN exposure, and the vesicle transport was disturbed, showing with aberrant expression and localization of Rab11a. Moreover, our results indicated that CTN exposure increased the expression of LAMP2, indicating the induction of lysosomal damage. In summary, our study showed that CTN exposure to mouse oocytes was toxic to the distribution and functions of organelles, which further led to a decrease of oocyte quality.

Actin filaments are widely involved in multiple cellular processes in oocyte meiosis, such as spindle migration and polar body extrusion. The actin nucleators like Arp2/3 complex and formins are the most recognized molecules for actin assembly in oocytes. In the present study, we report that the vesicle trafficking factor, RAB8A GTPase, is a new regulator critical for actin assembly in meiosis. Our results showed that RAB8A was localized at both the spindle periphery and cortex in mouse oocytes, which was similar to the localization patterns of actin filaments. RAB8A depletion caused spindle migration defects and the failure of polar body extrusion, which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes. Based on mass spectrometry analysis, we showed that RAB8A promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway. Moreover, we demonstrated that RAB8A colocalized and interacted with GM130 at the spindle periphery and that RAB8A depletion caused the disruption of GM130-docked Golgi distribution. Taken together, our data indicated that RAB8A was required for Golgi distribution, spindle migration, and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.