Chromatin Remodeling and Male Killing like spindles: these gonomeric spindles appear disorganized and the two spindles that hold the parental complements are distant from each other. (I) Arrested anaphase spindles: chromosomes are arrested at the metaphase plate and centrosomes detached from the poles (arrows). (J) Abnormal anaphase: chromosomes move in a disorderly fashion to the opposite poles of the spindle. (K) Abnormal telophase: the spindle lacks a distinct midzone and chromatin forms large bridges. (L) Irregular second mitosis: spindles are unusually elongated and the chromosomes are stretched among the opposite poles. Arrowheads point to remnant of the sperm tail; Pb, polar bodies. Scale bar is 10 mm. doi:10.1371/journal.pone.0030045.g001 cycles. To test this possibility we examined embryos collected 60– 90 minutes AED (Table S1). The main phenotypic class in this collection was represented by embryos found to be in the second mitosis (n = 46), as well as embryos in intravitelline mitoses (n = 149) (Fig. 2A,A9). However, further analysis revealed that some embryos that seemed to properly execute intravitelline mitoses had occasional defects consisting of spindles in which the progression through the nuclear division cycles was irregular. Defects such as barrel-shaped spindles with chromosomes in metaphase-like configuration (Fig. 2B,B9) and anaphase figures with lagging chromatids (Fig. 2C,C9) were encountered. Of the eggs scored 60–90 minutes AED, 16.7% (n = 45) displayed dramatic abnormalities consisting of barrel shaped spindles that held chromosomes mostly oriented in a metaphase-like configuration (Fig. 2D,D9). Isolated centrosomes or free astral arrays of microtu- bules were never observed at this developmental stage. These figures, presumably, resulted from an early arrest due to the failure of sperm entry, as suggested by the absence of a sperm tail. The remaining embryos scored 60–90 minutes AED (n = 30; 11.1%) displayed biastral or monastral bipolar spindles as well as barrel shaped spindles that often fused together in larger microtubule aggregates in which the chromatin was irregularly packed (Fig. 2E,E9). Figure 2. Improper chromosome segregation during the intravitelline mitoses. Black and white panels represent DNA staining alone; colour panels represent microtubules (green), DNA (red), and the sperm tail (blue). (A,A9) Detail of a normal embryo during early telophase of the seventh nuclear division cycle: sister chromatids have migrated at the opposite poles of the spindles to form daughter nuclei. Detail of embryos during early telophase (B,B9) or late anaphase (C,C9) of the seventh mitosis showing abnormal spindles: sister chromatids either do not separate and stay at the spindle equator (arrows in B) and the spindles are barrel-shaped (arrows in B9) or begin to separate, but do not migrate properly lagging in the spindle midzone or forming chromatin bridges (arrows in C), spindles do not show apparent defects at this stage (arrows in C9). (D,D9) Unfertilized eggs fixed 1 hour AED: note the anastral barrel spindles. (E,E9). Abnormal fertilized embryos fixed 1 hour AED: abnormal mitotic figures range from barrel-shaped biastral spindles (arrows in E9) to aberrant late telophase spindles in which the chromatin form dense bridges (larger arrows in E) and the centrosomes maintain their position closest to daughter nuclei (arrowheads in E9); late telophase spindles may be still recognized by the midbody remnants (small arrows in E9); double arrows in E9 point to the sperm tail. Bacteria associate with the poles of biastral spindles, but not with the poles of the anastral spindles. Scale bar is 4 mm. doi:10.1371/journal.pone.0030045.g002 PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30045