Sis in Drosophila [24] and human U2OS, HeLa and HaCaT cells [19]. Therefore, we examined the localization of ASPM in mouse oocytes at different stages by immunofluorescence. Our results showed that ASPM and the spindle microtubule protein Ac-tubulin were Sudan I chemical information colocalized to the entire spindle at MI and MII and also overlapped at the midbody in telophase I (Figure 1B). This localization pattern may indicate that ASPM regulates spindle assembly during mouse oocyte maturation. However, this 1655472 was inconsistent with theMorphological and Functional Study of ASPM GeneFigure 2. Localization of ASPM in mouse oocytes treated with spindle-perturbing agents. (A) MI oocytes were incubated with 20 mg/ml nocodazole for 5, 10 or 15 min and then costained with Ac-tubulin (green), ASPM (red) and DAPI (blue). Bar = 10 mm. (B) MI oocytes were incubated with 10 mM taxol for 45 min and then costained with Ac-tubulin (green), ASPM (red) and DAPI (blue). Bar = 10 mm. doi:10.1371/journal.pone.0049303.gpreviously published observations of ASPM localization during mitosis, so we investigated the localization in mouse MEFs to ensure the reliability of the ASPM antibody. In MEFs, ASPM localized to the spindle poles during mitotic metaphase (Figure 1C). Therefore, we deduced that ASPM played different roles in mitosis and meiosis. Meanwhile, this was the first demonstration of ASPM localization in mouse oocytes. The subcellular colocalization of Ac-tubulin and ASPM during meiotic maturation prompted us to further explore the relationship between microtubules and ASPM using the spindle-perturbing drugs nocodazole and taxol. After nocodazole treatment, microtubules were depolymerized; ASPM and Ac-tubulin disappeared from the spindle upon complete spindle collapse. After taxol treatment, microtubule fibers were excessively polymerized and formed a large spindle, together with numerous asters in the cytoplasm of treated oocytes, ASPM and Ac-tubulin colocalized to the spindle and cytoplasmic asters. Therefore, the localizationpattern of ASPM is similar to that of other proteins that are involved in spindle formation, such as septin1, which is most likely attached to the spindle components, perhaps Pleuromutilin chemical information participating in meiotic spindle assembly and maintenance [25]. To dissect the role of ASPM in mouse oocyte maturation, we used gene-specific morpholino injection to knock down ASPM protein expression and assess the loss of function phenotype. Western blot confirmed that the ASPM protein level was reduced by 49.14 after ASPM morpholino injection. Previous reports showed that some conserved proteins involved in the regulation of spindle organization and spindle assembly are relatively stable, such as LGN and p38a, but even moderate downregulation can cause functional effects [8,22]. After morpholino injection, a large proportion of ASPM-ablated oocytes exhibited spindle assembly defects. The predominant phenotypes included spindle elongation and disorganized spindle poles. In C. elegans, ASPM, as a novel LIN-5 binding partner, acts together with calmodulin to promoteMorphological and Functional Study of ASPM GeneFigure 3. ASPM expression was successfully reduced in oocytes by ASPM morpholinos. (A) Western blot of ASPM analysis revealed a 49.14 decrease in the ASPM expression level following ASPM morpholino injection relative to the uninjected control and control morpholino groups. The same blot revealed comparable levels of a-tubulin in all 3 groups. Each sample contains 100 oocytes.Sis in Drosophila [24] and human U2OS, HeLa and HaCaT cells [19]. Therefore, we examined the localization of ASPM in mouse oocytes at different stages by immunofluorescence. Our results showed that ASPM and the spindle microtubule protein Ac-tubulin were colocalized to the entire spindle at MI and MII and also overlapped at the midbody in telophase I (Figure 1B). This localization pattern may indicate that ASPM regulates spindle assembly during mouse oocyte maturation. However, this 1655472 was inconsistent with theMorphological and Functional Study of ASPM GeneFigure 2. Localization of ASPM in mouse oocytes treated with spindle-perturbing agents. (A) MI oocytes were incubated with 20 mg/ml nocodazole for 5, 10 or 15 min and then costained with Ac-tubulin (green), ASPM (red) and DAPI (blue). Bar = 10 mm. (B) MI oocytes were incubated with 10 mM taxol for 45 min and then costained with Ac-tubulin (green), ASPM (red) and DAPI (blue). Bar = 10 mm. doi:10.1371/journal.pone.0049303.gpreviously published observations of ASPM localization during mitosis, so we investigated the localization in mouse MEFs to ensure the reliability of the ASPM antibody. In MEFs, ASPM localized to the spindle poles during mitotic metaphase (Figure 1C). Therefore, we deduced that ASPM played different roles in mitosis and meiosis. Meanwhile, this was the first demonstration of ASPM localization in mouse oocytes. The subcellular colocalization of Ac-tubulin and ASPM during meiotic maturation prompted us to further explore the relationship between microtubules and ASPM using the spindle-perturbing drugs nocodazole and taxol. After nocodazole treatment, microtubules were depolymerized; ASPM and Ac-tubulin disappeared from the spindle upon complete spindle collapse. After taxol treatment, microtubule fibers were excessively polymerized and formed a large spindle, together with numerous asters in the cytoplasm of treated oocytes, ASPM and Ac-tubulin colocalized to the spindle and cytoplasmic asters. Therefore, the localizationpattern of ASPM is similar to that of other proteins that are involved in spindle formation, such as septin1, which is most likely attached to the spindle components, perhaps participating in meiotic spindle assembly and maintenance [25]. To dissect the role of ASPM in mouse oocyte maturation, we used gene-specific morpholino injection to knock down ASPM protein expression and assess the loss of function phenotype. Western blot confirmed that the ASPM protein level was reduced by 49.14 after ASPM morpholino injection. Previous reports showed that some conserved proteins involved in the regulation of spindle organization and spindle assembly are relatively stable, such as LGN and p38a, but even moderate downregulation can cause functional effects [8,22]. After morpholino injection, a large proportion of ASPM-ablated oocytes exhibited spindle assembly defects. The predominant phenotypes included spindle elongation and disorganized spindle poles. In C. elegans, ASPM, as a novel LIN-5 binding partner, acts together with calmodulin to promoteMorphological and Functional Study of ASPM GeneFigure 3. ASPM expression was successfully reduced in oocytes by ASPM morpholinos. (A) Western blot of ASPM analysis revealed a 49.14 decrease in the ASPM expression level following ASPM morpholino injection relative to the uninjected control and control morpholino groups. The same blot revealed comparable levels of a-tubulin in all 3 groups. Each sample contains 100 oocytes.
