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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.


Knockdown of mDia formins impairs oocyte maturation and spindle formation.A. The RNA levels of mDia1 and mDia2 in oocytes injected with dsRNAs against mDia1 and mDia2. The expression levels are expressed relative to the expression level in control oocytes treated with control dsRNA. B. Morphology and mDia2 localization in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. Red: mDia2; green: α-tubulin; blue: DNA. White arrows indicate mDia2 located at the spindle poles in control oocytes. C. Quantification of mDia2 in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. ***: significantly different from the negative control dsRNA-injected oocytes (p < 0.001); N.S.: not significant (p > 0.05). The boxes show the interquartile range; the whiskers show the 1.5 × the interquartile range; the line inside the box represents the median. D. Knockdown of mDia1, mDia2, or mDia1 + mDia2 decreases the oocyte maturation rate and increases the symmetric division rate. dsRNA against mDia1, mDia2, or a mixture of mDia1 and mDia2 were microinjected into immature oocytes along with control dsRNA and cells were cultured for 12 h after which their status of development was assessed. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter. E. Knockdown of mDia2 or mDia1 + mDia2 impairs spindle formation. Oocytes injected with mDia2, mDia1 + mDia2, or control dsRNA were incubated for 9 h and their spindle morphology was assessed after immunostaining for alpha-tubulin (green), chromatin (blue), and mDia2 (red). F. Percentage of oocytes with a normal spindle after injection with Dia2, mDia1 + mDia2, or control dsRNA. The filled bar represents the oocytes that had a spindle with normal morphology, while the white bar represents the oocytes that displayed normal chromosome alignment in spindle. Each experiment was performed in triplicate and the mean ratios of cells with normal spindles and chromosome alignment were plotted. Error bars indicate the standard error of the mean (SEM). ***: significantly different (p < 0.005); N.S.: not significant (p > 0.05).
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pone.0123438.g004: Knockdown of mDia formins impairs oocyte maturation and spindle formation.A. The RNA levels of mDia1 and mDia2 in oocytes injected with dsRNAs against mDia1 and mDia2. The expression levels are expressed relative to the expression level in control oocytes treated with control dsRNA. B. Morphology and mDia2 localization in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. Red: mDia2; green: α-tubulin; blue: DNA. White arrows indicate mDia2 located at the spindle poles in control oocytes. C. Quantification of mDia2 in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. ***: significantly different from the negative control dsRNA-injected oocytes (p < 0.001); N.S.: not significant (p > 0.05). The boxes show the interquartile range; the whiskers show the 1.5 × the interquartile range; the line inside the box represents the median. D. Knockdown of mDia1, mDia2, or mDia1 + mDia2 decreases the oocyte maturation rate and increases the symmetric division rate. dsRNA against mDia1, mDia2, or a mixture of mDia1 and mDia2 were microinjected into immature oocytes along with control dsRNA and cells were cultured for 12 h after which their status of development was assessed. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter. E. Knockdown of mDia2 or mDia1 + mDia2 impairs spindle formation. Oocytes injected with mDia2, mDia1 + mDia2, or control dsRNA were incubated for 9 h and their spindle morphology was assessed after immunostaining for alpha-tubulin (green), chromatin (blue), and mDia2 (red). F. Percentage of oocytes with a normal spindle after injection with Dia2, mDia1 + mDia2, or control dsRNA. The filled bar represents the oocytes that had a spindle with normal morphology, while the white bar represents the oocytes that displayed normal chromosome alignment in spindle. Each experiment was performed in triplicate and the mean ratios of cells with normal spindles and chromosome alignment were plotted. Error bars indicate the standard error of the mean (SEM). ***: significantly different (p < 0.005); N.S.: not significant (p > 0.05).

Mentions: To ablate the mDia family formins, we designed dsRNA against mouse mDia1 and mDia2. As shown in Fig. 4a, microinjection of oocytes with dsRNAs against mDia1 and mDia2 effectively decreased their mRNA levels to 39% and 26%, respectively, compared to those in the controls. We checked the protein levels of mDia2 using immunostaining and western blotting. As shown in Fig. 4b and 4c, the mDia2 protein level significantly decreased by mDia2 knockdown.


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)

Knockdown of mDia formins impairs oocyte maturation and spindle formation.A. The RNA levels of mDia1 and mDia2 in oocytes injected with dsRNAs against mDia1 and mDia2. The expression levels are expressed relative to the expression level in control oocytes treated with control dsRNA. B. Morphology and mDia2 localization in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. Red: mDia2; green: α-tubulin; blue: DNA. White arrows indicate mDia2 located at the spindle poles in control oocytes. C. Quantification of mDia2 in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. ***: significantly different from the negative control dsRNA-injected oocytes (p < 0.001); N.S.: not significant (p > 0.05). The boxes show the interquartile range; the whiskers show the 1.5 × the interquartile range; the line inside the box represents the median. D. Knockdown of mDia1, mDia2, or mDia1 + mDia2 decreases the oocyte maturation rate and increases the symmetric division rate. dsRNA against mDia1, mDia2, or a mixture of mDia1 and mDia2 were microinjected into immature oocytes along with control dsRNA and cells were cultured for 12 h after which their status of development was assessed. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter. E. Knockdown of mDia2 or mDia1 + mDia2 impairs spindle formation. Oocytes injected with mDia2, mDia1 + mDia2, or control dsRNA were incubated for 9 h and their spindle morphology was assessed after immunostaining for alpha-tubulin (green), chromatin (blue), and mDia2 (red). F. Percentage of oocytes with a normal spindle after injection with Dia2, mDia1 + mDia2, or control dsRNA. The filled bar represents the oocytes that had a spindle with normal morphology, while the white bar represents the oocytes that displayed normal chromosome alignment in spindle. Each experiment was performed in triplicate and the mean ratios of cells with normal spindles and chromosome alignment were plotted. Error bars indicate the standard error of the mean (SEM). ***: significantly different (p < 0.005); N.S.: not significant (p > 0.05).
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pone.0123438.g004: Knockdown of mDia formins impairs oocyte maturation and spindle formation.A. The RNA levels of mDia1 and mDia2 in oocytes injected with dsRNAs against mDia1 and mDia2. The expression levels are expressed relative to the expression level in control oocytes treated with control dsRNA. B. Morphology and mDia2 localization in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. Red: mDia2; green: α-tubulin; blue: DNA. White arrows indicate mDia2 located at the spindle poles in control oocytes. C. Quantification of mDia2 in oocytes injected with control, mDia2, or mDia1 + mDia2 dsRNAs. ***: significantly different from the negative control dsRNA-injected oocytes (p < 0.001); N.S.: not significant (p > 0.05). The boxes show the interquartile range; the whiskers show the 1.5 × the interquartile range; the line inside the box represents the median. D. Knockdown of mDia1, mDia2, or mDia1 + mDia2 decreases the oocyte maturation rate and increases the symmetric division rate. dsRNA against mDia1, mDia2, or a mixture of mDia1 and mDia2 were microinjected into immature oocytes along with control dsRNA and cells were cultured for 12 h after which their status of development was assessed. Significant differences (p < 0.05) between different treatments are indicated by a different superscript letter. E. Knockdown of mDia2 or mDia1 + mDia2 impairs spindle formation. Oocytes injected with mDia2, mDia1 + mDia2, or control dsRNA were incubated for 9 h and their spindle morphology was assessed after immunostaining for alpha-tubulin (green), chromatin (blue), and mDia2 (red). F. Percentage of oocytes with a normal spindle after injection with Dia2, mDia1 + mDia2, or control dsRNA. The filled bar represents the oocytes that had a spindle with normal morphology, while the white bar represents the oocytes that displayed normal chromosome alignment in spindle. Each experiment was performed in triplicate and the mean ratios of cells with normal spindles and chromosome alignment were plotted. Error bars indicate the standard error of the mean (SEM). ***: significantly different (p < 0.005); N.S.: not significant (p > 0.05).
Mentions: To ablate the mDia family formins, we designed dsRNA against mouse mDia1 and mDia2. As shown in Fig. 4a, microinjection of oocytes with dsRNAs against mDia1 and mDia2 effectively decreased their mRNA levels to 39% and 26%, respectively, compared to those in the controls. We checked the protein levels of mDia2 using immunostaining and western blotting. As shown in Fig. 4b and 4c, the mDia2 protein level significantly decreased by mDia2 knockdown.

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.