Expression and function of Dicer in adult spermatogenesis.

<p>(<b>A</b>) Domain structure of the Dicer protein is shown. The organization of the 5′ portion of <i>Dicer</i> locus is depicted. The targeting vector used for introduction of <i>FlagHA₂</i> into the <i>Dicer</i> locus and the schematic map of the targeted <i>Dicer</i> gene before and after Cre mediated-recombination are shown. Triangles represent <i>loxP</i> sites as indicated. Rectangles indicate the position of <i>Neomycin (Neo)</i> and <i>Diptheria toxin A (DTA)</i> selection marker genes. The SacI restriction sites are indicated as well as the respective Southern fragments detected by the 3′probe. A schematic diagram of the resulting FlagHA₂-Dicer protein is shown. (<b>B</b>) Southern blot of tail derived SacI-digested DNA from wild-type and Dcr^+/FH-Neo mice is shown with the 3′ probe indicated in A. (<b>C</b>) Western blot using anti-HA and anti-SMC1 antibodies on extracts from adult wild type and Dcr^+/FH testis is shown. (<b>D</b>) Immunofluorescence using anti-HA and anti-γH2AX antibodies on Dcr^+/FH testis germ cells from adult testis sections is shown. Scale bar = 10 µm. (<b>E</b>) Hematoxylin and eosin stained testis section from adult Dcr^Ctl and Dcr^C-KO mice with representative tubules shown. Scale bars = 50 µm and 20 µm in the upper and lower panel, respectively. (<b>F</b>) Increased apoptosis in Dcr^C-KO testis. A TUNEL assay counterstained with DAPI is shown on testis sections from adult Dcr^Ctl and Dcr^C-KO mice. The apoptotic cells stain in green. Scale bars = 50 µm and 10 µm in the upper and lower panel, respectively. Abbreviations: P, pachytene and RS, round spermatid. Representative images are shown from at least 3 mice analyzed in panels D–F.</p

miR-34bc/449 regulates a small cohort of genes in spermatocytes.

<p>(<b>A</b>) Expression scatterplot showing relative average expression of affymetrix probes between control (x-axis) and miR-34bc^−/−;449^−/− (y-axis). Significantly deregulated (p = 0.05) genes with a log₂ fold change of 0.5 (red) are shown. (<b>B</b>) The list of the 13 upregulated genes with predicted miR-34 seed binding sites is shown. Also indicated is the gene function as well as number of miR-34 binding sites. (<b>C</b>) qRT-PCR expression analysis of representative miR-34 family seed-containing deregulated genes identified. Normalized data are plotted as relative fold change in miR-34bc^−/−;449^−/− versus wild type pachytene spermatocytes. Standard error is shown and the asterisk indicates significantly upregulated expression (P<0.05). Other genes identified from the array that change in expression are also presented. The data in all panels are from biological quadruplicates of each genotype. (<b>D</b>) Sylamer enrichment landscape plot for all 876 7 nt words complementary to canonical mouse miRNA seed regions. The y-axis represents the sorted genelist of 21,560 genes from most up-regulated to most down-regulated in the miR-34bc^−/−;449^−/− pachytene spermatocytes. Each 7mer word was tested for significant enrichment across the 3′UTRs of genes in this list. The word corresponding to seed matching miR-34 family (Red) is enriched in the up-regulated genes.</p

miR-34b/c and miR-449 are selectively expressed in post-mitotic spermatogenesis.

<p>(<b>A</b>) Heat diagram summarizing the expression of miRNAs in mitotic <i>in vitro</i> cultured spermatogonial stem cell (SSC) lines, <i>ex vivo</i> isolated meiotic spermatocytes (sp.cytes) and spermiogenic round spermatids (RS). The average expression of biological replicates is shown. (<b>B</b>) The mature sequence of the murine miR-34 family miRNAs is shown with the seed sequence highlighted in red. The expression of the miR-34 family members is summarized from the array data. (<b>C</b>) The expression of miR-34 family was determined by Northern blotting of RNA derived from a broad panel of tissues. U6 snRNA and Let-7a was used as loading controls. Abbreviations: BM, bone marrow; Sp, spleen; Thy, Thymus; Lym, lymph node; Te, testis; Ov, ovary; Mu, muscle, He, heart; Li, liver; Lu, lung; Kid. Kidney and Pe, peritoneal cavity cells. (<b>D</b>) Expression of miR-34 family members was determined in the first wave of spermatogenesis by Northern blotting of total RNA from testis at the indicated day post partum (dpp), Ad indicates adult. U6 snRNA was used as a loading control. Representative data is shown from two independent experiments in panel C and D. (<b>E</b>) The onset of miR-34c expression in early pachytene spermatocytes during the first wave of spermatogenesis. The spatial expression of miR-34c (Green) is shown by <i>in situ</i> hybridization on sections of 14 dpp mouse testis, the section were counterstained with anti-γH2AX (Red) antibodies to precisely identify the meiotic stage. Scale bar = 30 µm. Abbreviations: L, leptotene; Z, zygotene; eP, early pachytene and P, pachytene. (<b>F</b>) The expression of miR-34c (Green) by <i>in situ</i> hybridization in the indicated adult spermatogenic populations is shown. Anti-γH2AX (Red) was used as in (E). Scale bar = 10 µm. Representative images from one of three independent experiments are shown for panel E and F.</p

miR-34bc/449 are required for multiple stages of post-mitotic spermatogenesis.

<p>(<b>A</b>) PAS stained testis section from adult control and miR-34bc^−/−;449^−/− mice is shown. Overview of several tubules is shown in upper panels. Magnified and staged tubules are presented in the lower panels, the schematic diagram summarizes the spermatogenic content of tubules in wild type mice. Abbreviations: Pl, preleptotene; Z, zygotene; P, pachytene; D, diplotene;; RS, round spermatid and ES, elongating spermatid. Scale bars = 50 µm and 10 µm in the upper and lower panels, respectively. (<b>B</b>) MiR-34bc^−/−;449^−/− testis sections and percentages of tubules with meiotic arrest at zygotene stage is shown. Scale bars = 30 µm and 20 µm in the upper and lower panel, respectively. Representative images are shown from 6 mice analysed is shown in panel A and B. (<b>C</b>) Increased apoptosis in miR-34bc^−/−;449^−/− testis. A TUNEL assay counterstained with DAPI (Blue) is shown on testis sections from adult control and miR-34bc^−/−;449^−/− mice (upper panel). The apoptotic cells stain in green. Apoptotic miR-34bc^−/−;449^−/− pachytene (P) and elongating spermatid (ES) are shown in the lower panel along with non-apoptotic control cells. Scale bars = 30 µm and 10 µm in the upper and lower panel, respectively. Representative images are shown from 3 mice analysed is shown. (<b>D</b>) FACS plot of adult testis shown, gated populations in upper panel I (lepto-zygotene) and II (pachytene-diplotene), in lower panel III (round spermatids) and IV (elongating spermatids). Numbers indicated the overall percentage of the respective populations. (<b>E</b>) Comparative enumeration of spermatogenic populations of control (Ctl) and miR-34bc/499^−/− mice. Total testicular cell numbers (upper) are shown. Numbers plotted for the developmentally defined subpopulations indicated in (D) by roman numerals (lower panel). 8 animals per genotype were analyzed by FACS. Mean ±s.e.m. values are shown in the graph. (<b>F</b>) Schematic diagram indicating the expression and impact of loss of miR-34bc/449 expression. * and *** indicates a <i>p</i> value (unpaired t test with Welch correction) of <0.05 and <0.001 respectively.</p