Heat map representing expression levels of 77 currently known X-linked miRNAs in 12 organs of adult mice. Expression levels were determined by semi-quantitative PCR using small RNA cDNAs.

Heat map representing expression levels of 77 X-linked miRNAs in developing testes and 9 purified spermatogenic cell populations. P7-21, postnatal days 7–21; Ad, adult; SgPr, primitive type A spermatogonia; SgA, type A spermatogonia; SgB, type B spermatogonia; SpPr, preleptotene spermatocytes; SpLZ, leptotene and zygotene spermatocytes; SpPaJ, pachytene spermatocytes from juvenile mice at postnatal day 18; SpPaA, pachytene spermatocytes from adult mice; SdR, round spermatids; SdE, elongated spermatids and residual bodies. Underlined X-linked miRNA genes also have one or more autosomal copies and thus the expression detected may be derived from the × chromosome and /or their autosomal copies.

Three types of X-linked miRNAs grouped according to their expression patterns during spermatogenesis. Type I X-linked miRNAs are expressed in premeiotic spermatogonia followed by decreased expression in spermatocytes and spermatids, indicating that they are subject to MSCI and PMSC. Type II X-linked miRNAs show increased expression in spermatocytes indicating escape from MSCI, followed by decreased expression in spermatids indicating that they are subject to PMSC. Type III X-linked miRNAs display elevated expression in both spermatocytes and spermatids indicating escape from both MSCI and PMSC. SgPr, primitive type A spermatogonia; SgA, type A spermatogonia; SgB, type B spermatogonia; SpPr, preleptotene spermatocytes; SpLZ, leptotene and zygotene spermatocytes; SpPaJ, pachytene spermatocytes from juvenile mice at postnatal day 18; SpPaA, pachytene spermatocytes from adult mice; SdR, round spermatids; SdE, elongated spermatids and residual bodies; Sz, spermatozoa.

Localization of three X-linked miRNAs in adult mouse testes using locked nucleic acid-based miRNA in situ hybridization. (a) Hybridization signals (blue) for mir-718-3p in stage VIII seminiferous tubule section. (b) Immunofluorescence staining of γH2AX (green) as a marker for spermatocytes in the same section shown in a. (c) Propidium iodide (PI) staining (red) of cell nuclei of the same section shown in a. (d) Merge of a, b and c. (e) Hybridization signals (blue) for mir-883a-3p in stage V (left) and stage IX (right) seminiferous tubule sections. (f) Immunofluorescence staining of γH2AX (green) in the same section shown in e. (g) PI staining of cell nuclei (red) of the same section shown in e. (h) Merge of e, f and g. (i) High-power image of the framed area in h. (j) Hybridization signals (blue) for mir-883a-5p in stage VI seminiferous tubule section. (k) Immunofluorescence staining of mouse MIWI (green) as a marker for round spermatids in the same section shown in j. (l) PI staining of cell nuclei in the same section shown in j. (m) Merge of j, k and l. (n) High-power image of the framed area in m. Scale bars= 10µm. Panels a–d, e–h and j–m are shown at the same magnifications, respectively. S, sex body (also called XY body); CB, chromatoid body; Sg, spermatogonia; Pl, preleptotene spermatocytes; L, leptotene spermatocytes; P, pachytene spermatocytes; Sd5, 8, 15, step 5, 8 and 15spermatids, respectively.

ChIP-qPCR analyses of interactions between RNA polymerase II (pol II) and three X-linked miRNA genes. (a) Formaldehyde cross-linked genomic DNAs from postnatal day 7 (P7), P10, P14, P17, P21 and adult testes were reduced to fragment sizes of 180 bp to 330 bp via enzymatic digestion. (b) qPCR analyses of levels of Pgk1, Pgk2, Mirn743a, Mirn883a and Mirn106a in RNA pol II ChIP products from developing mouse testes. Values are presented as means ±SEM, n=3. Significantly different values are marked with *( p ≤ 0.05) or ** (p ≤ 0.01). (c) Analysis of qPCR products on 2% agarose gels.