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Small molecule inhibitor of formin homology 2 domains (SMIFH2) reveals the roles of the formin family of proteins in spindle assembly and asymmetric division in mouse oocytes.

Kim HC, Jo YJ, Kim NH, Namgoong S - PLoS ONE (2015)

Bottom Line: Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels.In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation.Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, Chungbuk National University, Cheong-Ju, ChungBuk, Republic of Korea.

ABSTRACT
Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.

No MeSH data available.


Treatment with SMIFH2 decreases the actin level in oocytes and impairs spindle formation.A. Treatment with SMIFH2 decreases the cortical actin level in maturing mouse oocytes. Control (left) and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Chromatin was stained with Hoechst 33342 (blue). Bar = 10 μm. B. Quantification of the cortical actin levels in SMIFH2-treated (300 and 500 μM) and control oocytes. Actin in the cortex regions excluding the cortical actin cap and polar body regions was quantified using ImageJ[42]. The box represents the interquartile range; the whiskers show 1.5x the interquartile range; line inside the box represents the median (control: n = 14; 300 μM: n = 12; 500 μM: n = 12). N.S.: not significant (p > 0.05); ***: significantly different from control oocytes (p < 0.001). C. Phalloidin-stained cytoplasmic actin mesh in oocytes. Untreated (control) oocytes and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Bar = 3 μm. D. Quantification of the cytoplasmic actin stained with phalloidin in control (n = 27) and SMIFH2-treated (300 μM: n = 21; 500 μM: n = 7) cells. E. Abnormal spindle formation induced by SMIFH2 treatment. Spindles of control or SMIFH2-treated (300 and 500 μM) oocytes were visualized by immunostaining with anti-α-tubulin (green), and chromatin was stained with Hoechst 33342 (blue). No spindle structures are detected in oocytes treated with 500 μM of SMIFH2. Bar: 10 μm. F. Effect of various SMIFH2 concentrations on spindle formation and morphology. Oocytes were classified based on the spindle morphology. Normal: oocytes with normal spindle shape with proper chromatin alignment, similar to the spindle shape of the control cells in panel E; Abnormal: oocytes containing a spindle with improper chromatin alignment or irregular shape, similar to that of cells treated with 300 μM SMIFH2 in panel E; No spindle: oocytes with no detectable spindle structure, similar to that of cells treated with 500 μM SMIFH2. The ratio of each spindle type of oocytes to total oocytes was plotted. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter(a,b and c).
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pone.0123438.g002: Treatment with SMIFH2 decreases the actin level in oocytes and impairs spindle formation.A. Treatment with SMIFH2 decreases the cortical actin level in maturing mouse oocytes. Control (left) and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Chromatin was stained with Hoechst 33342 (blue). Bar = 10 μm. B. Quantification of the cortical actin levels in SMIFH2-treated (300 and 500 μM) and control oocytes. Actin in the cortex regions excluding the cortical actin cap and polar body regions was quantified using ImageJ[42]. The box represents the interquartile range; the whiskers show 1.5x the interquartile range; line inside the box represents the median (control: n = 14; 300 μM: n = 12; 500 μM: n = 12). N.S.: not significant (p > 0.05); ***: significantly different from control oocytes (p < 0.001). C. Phalloidin-stained cytoplasmic actin mesh in oocytes. Untreated (control) oocytes and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Bar = 3 μm. D. Quantification of the cytoplasmic actin stained with phalloidin in control (n = 27) and SMIFH2-treated (300 μM: n = 21; 500 μM: n = 7) cells. E. Abnormal spindle formation induced by SMIFH2 treatment. Spindles of control or SMIFH2-treated (300 and 500 μM) oocytes were visualized by immunostaining with anti-α-tubulin (green), and chromatin was stained with Hoechst 33342 (blue). No spindle structures are detected in oocytes treated with 500 μM of SMIFH2. Bar: 10 μm. F. Effect of various SMIFH2 concentrations on spindle formation and morphology. Oocytes were classified based on the spindle morphology. Normal: oocytes with normal spindle shape with proper chromatin alignment, similar to the spindle shape of the control cells in panel E; Abnormal: oocytes containing a spindle with improper chromatin alignment or irregular shape, similar to that of cells treated with 300 μM SMIFH2 in panel E; No spindle: oocytes with no detectable spindle structure, similar to that of cells treated with 500 μM SMIFH2. The ratio of each spindle type of oocytes to total oocytes was plotted. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter(a,b and c).

Mentions: To investigate the exact mechanism of SMIFH2-mediated inhibition of oocyte maturation, we examined the distribution of the actin filaments and microtubules in SMIFH2-treated and control oocytes by using immunostaining. As shown in Fig. 2A and 2B, the amount of cortical actin decreased significantly in the SMIFH2-treated groups, confirming that formins play a role in establishing the cortical actin in oocytes. In addition, we measured the amount of actin within the cytoplasmic actin meshwork in control and SMIFH2-treated cells, which showed that SMIFH2 effectively decreased the actin mesh levels (Fig. 2C and 2D), confirmed the previous reports[10, 28, 32] that cytoplasmic actin mesh in oocytes was generated by formin-2.


Small molecule inhibitor of formin homology 2 domains (SMIFH2) reveals the roles of the formin family of proteins in spindle assembly and asymmetric division in mouse oocytes.

Kim HC, Jo YJ, Kim NH, Namgoong S - PLoS ONE (2015)

Treatment with SMIFH2 decreases the actin level in oocytes and impairs spindle formation.A. Treatment with SMIFH2 decreases the cortical actin level in maturing mouse oocytes. Control (left) and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Chromatin was stained with Hoechst 33342 (blue). Bar = 10 μm. B. Quantification of the cortical actin levels in SMIFH2-treated (300 and 500 μM) and control oocytes. Actin in the cortex regions excluding the cortical actin cap and polar body regions was quantified using ImageJ[42]. The box represents the interquartile range; the whiskers show 1.5x the interquartile range; line inside the box represents the median (control: n = 14; 300 μM: n = 12; 500 μM: n = 12). N.S.: not significant (p > 0.05); ***: significantly different from control oocytes (p < 0.001). C. Phalloidin-stained cytoplasmic actin mesh in oocytes. Untreated (control) oocytes and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Bar = 3 μm. D. Quantification of the cytoplasmic actin stained with phalloidin in control (n = 27) and SMIFH2-treated (300 μM: n = 21; 500 μM: n = 7) cells. E. Abnormal spindle formation induced by SMIFH2 treatment. Spindles of control or SMIFH2-treated (300 and 500 μM) oocytes were visualized by immunostaining with anti-α-tubulin (green), and chromatin was stained with Hoechst 33342 (blue). No spindle structures are detected in oocytes treated with 500 μM of SMIFH2. Bar: 10 μm. F. Effect of various SMIFH2 concentrations on spindle formation and morphology. Oocytes were classified based on the spindle morphology. Normal: oocytes with normal spindle shape with proper chromatin alignment, similar to the spindle shape of the control cells in panel E; Abnormal: oocytes containing a spindle with improper chromatin alignment or irregular shape, similar to that of cells treated with 300 μM SMIFH2 in panel E; No spindle: oocytes with no detectable spindle structure, similar to that of cells treated with 500 μM SMIFH2. The ratio of each spindle type of oocytes to total oocytes was plotted. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter(a,b and c).
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pone.0123438.g002: Treatment with SMIFH2 decreases the actin level in oocytes and impairs spindle formation.A. Treatment with SMIFH2 decreases the cortical actin level in maturing mouse oocytes. Control (left) and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Chromatin was stained with Hoechst 33342 (blue). Bar = 10 μm. B. Quantification of the cortical actin levels in SMIFH2-treated (300 and 500 μM) and control oocytes. Actin in the cortex regions excluding the cortical actin cap and polar body regions was quantified using ImageJ[42]. The box represents the interquartile range; the whiskers show 1.5x the interquartile range; line inside the box represents the median (control: n = 14; 300 μM: n = 12; 500 μM: n = 12). N.S.: not significant (p > 0.05); ***: significantly different from control oocytes (p < 0.001). C. Phalloidin-stained cytoplasmic actin mesh in oocytes. Untreated (control) oocytes and oocytes treated with 500 μM of SMIFH2 were stained with phalloidin to visualize actin (red). Bar = 3 μm. D. Quantification of the cytoplasmic actin stained with phalloidin in control (n = 27) and SMIFH2-treated (300 μM: n = 21; 500 μM: n = 7) cells. E. Abnormal spindle formation induced by SMIFH2 treatment. Spindles of control or SMIFH2-treated (300 and 500 μM) oocytes were visualized by immunostaining with anti-α-tubulin (green), and chromatin was stained with Hoechst 33342 (blue). No spindle structures are detected in oocytes treated with 500 μM of SMIFH2. Bar: 10 μm. F. Effect of various SMIFH2 concentrations on spindle formation and morphology. Oocytes were classified based on the spindle morphology. Normal: oocytes with normal spindle shape with proper chromatin alignment, similar to the spindle shape of the control cells in panel E; Abnormal: oocytes containing a spindle with improper chromatin alignment or irregular shape, similar to that of cells treated with 300 μM SMIFH2 in panel E; No spindle: oocytes with no detectable spindle structure, similar to that of cells treated with 500 μM SMIFH2. The ratio of each spindle type of oocytes to total oocytes was plotted. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter(a,b and c).
Mentions: To investigate the exact mechanism of SMIFH2-mediated inhibition of oocyte maturation, we examined the distribution of the actin filaments and microtubules in SMIFH2-treated and control oocytes by using immunostaining. As shown in Fig. 2A and 2B, the amount of cortical actin decreased significantly in the SMIFH2-treated groups, confirming that formins play a role in establishing the cortical actin in oocytes. In addition, we measured the amount of actin within the cytoplasmic actin meshwork in control and SMIFH2-treated cells, which showed that SMIFH2 effectively decreased the actin mesh levels (Fig. 2C and 2D), confirmed the previous reports[10, 28, 32] that cytoplasmic actin mesh in oocytes was generated by formin-2.

Bottom Line: Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels.In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation.Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, Chungbuk National University, Cheong-Ju, ChungBuk, Republic of Korea.

ABSTRACT
Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.

No MeSH data available.