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Background: Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation. Results: Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion. Conclusions: In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes. [ABSTRACT FROM AUTHOR]

Mammalian diaphanous1 (mDia1) is a homologue of Drosophila diaphanous and belongs to the Formin-homology family of proteins that catalyze actin nucleation and polymerization. Although Formin family proteins, such as Drosophila diaphanous, have been shown to be essential for cytokinesis, whether and how mDia1 functions during meiosis remain uncertain. In this study, we explored possible roles and the signaling pathway involved for mDia1 using a mouse oocyte model. mDia1 depletion reduced polar body extrusion, which may have been due to reduced cortical actin assembly. mDia1 and Profilin1 had similar localization patterns in mouse oocytes and mDia1 knockdown resulted in reduced Profilin1 expression. Depleting FMNL1, another Formin family member, resulted in reduced mDia1 expression, while RhoA inhibition did not alter mDia1 expression, which indicated that there was a FMNL1-mDia1-Profilin1 signaling pathway in mouse oocytes. Additionally, mDia1 knockdown resulted in disrupting oocyte spindle morphology, which was confirmed by aberrant p-MAPK localization. Thus, these results demonstrated indispensable roles for mDia1 in regulating mouse oocyte meiotic maturation through its effects on actin assembly and spindle organization. [ABSTRACT FROM AUTHOR]

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development. [ABSTRACT FROM AUTHOR]

Abstract: The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning. [Copyright &y& Elsevier]

Background information During meiosis, a bipolar spindle forms in the central cytoplasm of an oocyte and then moves to the cortex to extrude the first polar body. This is dependent on the regulation of actin and actin-related molecules. Dynamin 2, a large guanosine triphosphatases (GTPase) known to regulate clathrin-mediated endocytosis, is involved in actin recruitment and actin-based vesicle mobility. In this study, we investigated the role of Dynamin 2 in oocyte meiosis. Results Dynamin 2 was localised at the cortex and around the spindles of oocytes. Disrupting Dynamin 2 activity by RNAi or an inhibitor resulted in polar body extrusion failure. Using time-lapse microscopy to monitor aberrant oocyte cytokinesis, the chromosomes were first separated, but then re-joined. Actin expression in oocytes was decreased; and actin cap formation was disrupted, which was confirmed by the disappearance of cortical-granule-free domains. In addition, live cell imaging showed that spindle migration had failed and that spindles were arrested centrally in oocytes. This may have been due to the Dynamin-binding protein Profilin and actin-related protein 2/3 (ARP2/3) complexes, which exhibited dispersed signals after disrupting Dynamin 2 activity. Conclusions Thus, our results indicate that Dynamin 2 regulates spindle migration and polar body extrusion during mouse oocyte meiosis through an actin-based pathway. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of various durations of dibutyryl cyclic AMP (dbcAMP) treatment on the in vitro maturation (IVM) and subsequent development of parthenogenetically activated embryos. Immature porcine oocytes were cultured with or without 1 mM dbcAMP during the first 20, 28, or 36 h of culture, and then incubated for an additional 24 h without dbcAMP. The expression ofWee1B,Myt, andCdc25Band the level of maturation promoting factor (MPF) in metaphase II oocytes were analyzed by real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The distribution of actin microfilaments in oocytes was also assessed. Subsequently, apoptotic cells in blastocysts from each group were visualized by transferase-mediated dUTP nick-end labeling staining. Results showed that oocytes extruded the first polar body between 12 and 18 h after being released from dbcAMP. MPF activity in oocytes at 28 + 24 h and 36 + 24 h after dbcAMP treatment was higher than that in the control group. Significantly more blastocysts were present among embryos in 28 + 24 h (54.28% vs. 39.11%,P< 0.05) and 36 + 24 h (47.24% vs. 32.94%,P< 0.05) groups than among embryos cultured in the absence of dbcAMP. However, the number of total and apoptotic cells was not significantly different between groups. The distribution of actin microfilaments was abnormal in oocytes cultured for 60 h without dbcAMP. In addition, the expression ofWee1B,Myt, andCdc25Bwas higher in the control group at 44 h than in the dbcAMP group, but there were no differences in expression at the other time points. In conclusion, dbcAMP treatment delays oocyte maturation and maintains oocyte quality. [ABSTRACT FROM AUTHOR]

The Arp2/3 complex regulates actin nucleation, which is critical for a wide range of cellular processes, such as cell polarity, cell locomotion, and endocytosis. In the present study, we investigated the possible roles of the Arp2/3 complex in porcine oocytes during meiotic maturation. Immunofluorescent staining showed the Arp2/3 complex to localize mainly to the cortex of porcine oocytes, colocalizing with actin. Treatment with an Arp2/3 complex specific inhibitor, CK666, resulted in a decrease in Arp2/3 complex localization at the oocyte cortex. The maturation rate of porcine oocytes decreased significantly after CK666 treatment, concomitant with the failure of cumulus cell expansion and oocyte polar body extrusion. The fluorescence intensity of F-actin decreased in the cytoplasm, and CK666 also disrupted actin cap formation. In summary, our results illustrate that the Arp2/3 complex is required for the meiotic maturation of porcine oocytes and that actin nucleation is critical for meiotic maturation. [ABSTRACT FROM AUTHOR]

Objective: To investigate the molecular mechanism of mouse oocyte polarity loss during aging. Design: Experimental study. Setting: Academic basic research laboratory. Animal(s): Mice. Intervention(s): Oocytes were collected 16 hours after injection of hCG and cultured in M16 medium for an additional 14 hours with or without caffeine. Main Outcome Measure(s): Expression and localizations of actin nucleators actin-related protein 2/3 complex, JMY, and WAVE2 were examined by immunofluorescence staining, and their messenger RNA levels were examined by real-time reverse transcription–polymerase chain reaction. Result(s): The protein and messenger RNA levels of actin-related protein 2/3 complex, JMY, and WAVE2 were decreased in aged oocytes, but the levels were normal in caffeine-treated aged oocytes. Conclusion(s): Our data indicated that the loss of oocyte polarity may be due to the degradation of actin nucleators in aged oocytes. [Copyright &y& Elsevier]

PLK4, a polo-like kinase (PLK) family member that accumulates in the cytoplasm, has been identified as a crucial regulator of centriole formation. PLK4 also controls several essential cellular functions, including cytokinesis and gene expression. In this study, we investigated the expression and function of PLK4 during bovine oocyte meiotic maturation and subsequent embryo development. The PLK4 mRNA was detected in bovine oocytes at all developmental stages during meiotic maturation. Immunofluorescence staining showed that PLK4 protein exhibited a dynamic localization pattern in the oocyte cytoplasm during meiotic maturation, and fluorescence immunostaining markedly increased in metaphase II. When an interfering double-stranded RNA targeting PLK4 was injected into germinal vesicle–stage oocytes, PLK4 transcript levels decreased significantly in metaphase II oocytes (P < 0.05). The PLK4 knockdown caused spindle defects and chromosome misalignment and considerably reduced the amount of cortical and cytoplasmic actin. PLK4 was localized in the cytoplasm of early embryos, and PLK4 knockdown in germinal vesicle–stage oocytes led to failure in the early development of in vitro fertilized embryos (P < 0.05). Taken together, these results indicated that PLK4 plays crucial roles in bovine oocyte meiotic maturation and subsequent early embryo development. [ABSTRACT FROM AUTHOR]