Figure 3 A Combined immunofluorescence and DNA FISH analysis of the kinetochore inner plate/centromere formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) spermatocytes at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1A. Chromosomes were stained with anti-centromere protein antibody (CREST, purple signal), indicating the kinetochore inner plate/centromere. Major satellite DNA regions were detected using an antisense probe (red signal) which revealed the specific localization of pericentromeres in chromosomes. Nuclear DNA was stained by DAPI (blue). Scale bars represent 2 µm.  B Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm. C Population of overlapping, adjacent, and separated inner kinetochores shown in B. The results represent the mean ± SD of three independent experiments. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 88, 118 and 125, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,520, 4,720 and 5,000, respectively.  D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm. E Magnified images of categories of sister kinetochore configurations from D. One arrow indicates overlapping pair. Double arrows indicate split pair. The scale bar represents 1 µm. F Population of the sister kinetochores shown in E. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 93, 89 and 81, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,720, 3,560 and 3,240, respectively.