Figure 2A Equal amounts of total RNA isolated from Miwi knockout (-/-), heterozygote (+/-) and slicer activity-deficient mutant (-/ADH) spermatocytes, were resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite RNA transcripts. The blot was re-hybridized with a 28S-complementary probe to demonstrate equal loading (bottom on the left). The relative abundance was quantified using ImageJ software, normalized to the 28S signal from the blot. B 100ng of total RNA was isolated from Miwi heterozygous (+/-), knockout (-/-) and slicer activity-deficient mutant (-/ADH) spermatocytes and was subjected to qRT-PCR with primers specific to major satellite RNA, minor satellite RNA, 5S, 5.8SSertoli-spermatogenic cell co-cultures were transfected with vectors ectopically expressing both strands of major satellite RNA (Maj, dark gray bar), both strands of minor satellite RNA (Min, black bar), full-length 18S (18S, light gray bar) or empty vector (Ctrl, white bar). Total RNA isolated from equal numbers of FACS-sorted MIWI-expressing spermatocytes were subjected to qRT-PCR with primers specific to major satellite RNA (C), minor satellite RNA (D), 18S (E) Sertoli-spermatogenic cell co-cultures were transfected with vectors ectopically expressing both strands of major satellite RNA (Maj, dark gray bar), both strands of minor satellite RNA (Min, black bar), full-length 18S (18S, light gray bar) or empty vector (Ctrl, white bar). Total RNA isolated from equal numbers of FACS-sorted MIWI-expressing spermatocytes were subjected to qRT-PCR with primers specific to 5.8S (F) and Gapdh (G). Data presented were normalized to 5S rRNA. 5.8S rRNA (F) and Gapdh (G) were used as negative controls. H Immunofluorescence images of Meiotic metaphase I plate in Sertoli-spermatogenic cell co-cultures. Shown were spermatocytes over-expressing both strands of major satellite RNA (Maj), both strands of minor satellite RNA (Min), full-length 18S (18S) or empty vector (Ctrl). Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bar, 5 μm. I Frequency of mis-arrangement of the meiotic metaphase I plate in Sertoli-spermatogenic cell cocultures using Miwi+/-;Stra8-Cre testes. Spermatocytes transfected with vectors expressing both strands of major satellite RNA (Maj, dark gray), both strands of minor satellite RNA (Min, black), full-length 18S (18S, light gray) or empty vector (Ctrl, white). n=3 cultures for each group. The total numbers of counted meiotic metaphase plate in Ctrl, 18S, Maj and Min were 153, 144, 130 and 141, respectively. J Combined RNA-DNA FISH analysis of major satellite transcripts and DNA regions in Miwi heterozygote (Miwi+/-) and knockout (Miwi-/-) diakinesis spermatocytes. Sense major satellite RNA transcripts were first detected using an antisense probe (red signal), and then the major satellite DNA region was further detected using a sense probe (green signal). For RNase A treatment, the slides were incubated with (+RNase A) or without (-RNase A) 100 μg/ml RNase A for 1 hour before adding antisense probe. Nuclear DNA was stained by DAPI (blue). Arrows indicate the same pericentric heterochromatin in each channel per cell. Scale bars represent 2 µm.