Effects of different carcass preservation temperatures on oocyte meiotic process.

<p>(A) The number of GV oocytes collected from mouse carcass after preserved for 4 hours at different temperatures. (B) The percentage of GVBD oocytes and PBE in PMI 4 h group at different preservation temperatures. Each bar represents mean ±SEM (n = 3). * P<0.05.</p

Effects of preservation time and temperature on mitochondrial distribution and GSH level in mouse oocytes.

<p>(A) Mitochondrial distribution was evaluated by fluorescence microscopy. Three localization patterns were observed: perinuclear, homogenous and clustered. (B) The percentage of abnormal mitochondrial distribution in GV oocytes in the control group (n = 279) and preservation group at different temperatures. * indicate statistically significant differences (P<0.05). (C) The GSH concentrations in GV oocytes (n = 30) in different groups.</p

Effects of different preservation periods and temperatures on oocyte spindle configuration after IVM.

<p>Oocytes cultured in M2 medium for 8h (MI) and 12h (MII), spindle was stained with FITC-α-tubulin and DNA was stained with PI. (A, B,C) the normal spindle in mouse MI and MII oocyte in control group and experimental group at different preservation temperatures.</p

RhoA-mediated FMNL1 regulates GM130 for actin assembly and phosphorylates MAPK for spindle formation in mouse oocyte meiosis

<p>Formin-like 1 (FMNL1) is a member of Formin family proteins which are the actin nucleators. Although FMNL1 activities have been shown to be essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis, the functional roles of mammalian FMNL1 during oocyte meiosis remain uncertain. In this study, we investigated the functions of FMNL1 in mouse oocytes using specific morpholino (MO) microinjection and live cell imaging. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD). FMNL1 knockdown caused the low rate of polar body extrusion and resulted in large polar bodies. Time-lapse microscopic and immunofluorescence intensity analysis indicated that this might be due to the aberrant actin expression levels. Cortical polarity was disrupted as shown by a loss of actin cap and cortical granule free domain (CGFD) formation, which was confirmed by a failure of meiotic spindle positioning. And this might be the reason for the large polar body formation. Spindle formation was also disrupted, which might be due to the abnormal localization of p-MAPK. These results indicated that FMNL1 affected both actin dynamics and spindle formation for the oocyte polar body extrusion. Moreover, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130. Finally, we found that the small GTPase RhoA might be the upstream regulator of FMNL1. Taken together, our data indicate that FMNL1 is required for spindle organization and actin assembly through a RhoA-FMNL1-GM130 pathway during mouse oocyte meiosis.</p