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Germ cell expression of the transcriptional co-repressor TIF1ß is required for the maintenance of spermatogenesis in the mouse

Philipp Weber*,{dagger}, Florence Cammas{dagger}, Christelle Gerard, Daniel Metzger, Pierre Chambon, Régine Losson{ddagger} and Manuel Mark{ddagger}

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP163, 67404 Illkirch-cedex, France
* Present address: Brain Research Institute, University and ETH Zurich, Winterthurerstrasse 190, CH 8057 Zurich, Switzerland
{dagger} These authors contributed equally to this work



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  		Fig. 1. Immunolocalization of TIF1ß in post-pubertal wild-type testis. Frozen histological sections were incubated with the anti-TIF1ß antibody whose binding to cell structures was then revealed with a Cy3-conjugated secondary antibody (red signal) and nuclei were counterstained with DAPI (blue signal). (A,D,G,J) Cy3 and (B,E,H,K) DAPI labeling.(C,F,I,L) Superimposition of the two fluorochromes. (G-L) Single optical sections of confocal microscopic analysis. Roman numerals refer to stages of the seminiferous epithelium cycle (Russell et al., 1990Go). Each stage is defined by a specific association of germ cell types. The cycle corresponds to the series of changes occurring at a given level of the seminiferous tubule between two successive appearances of the same cell association. In normal mice, there are 12 stages, designated I to XII, each corresponding to one of the first 12 steps of spermatid maturation. ES-9 and ES-13 correspond to steps in spermatid maturation. Ch, meiotic chromosomes; L, leptotene spermatocytes; P, pachytene spermatocytes; Nu, nucleolus of Sertoli cell; RS, round spermatids; S, Sertoli cells; Sa, satellite nucleolar heterochromatin of the Sertoli cell. The arrowheads and arrows in A-F indicate heterochromatin- and chromosome-associated TIF1ß, respectively. The broken lines indicate the contours of seminiferous tubules. Scale bar: 30 µm in A-C; 15 µm in D-F; 3 µm in G-L.

 


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  		Fig. 2. Immunolocalization of TIF1ß in post-pubertal wild-type testis. Histological sections from paraffin wax-embedded testes were incubated with the anti-TIF1ß antibody, whose binding to cell structures was then revealed with a Cy3-conjugated secondary antibody (red signal) and nuclei were counterstained with DAPI (blue signal). BrdU incorporation in the tubular cross section shown in C was detected using a green fluorochrome. Roman numerals refer to stages of the seminiferous epithelium cycle. ES-10 and ES-16 correspond to steps in spermatid maturation. L, leptotene spermatocytes; LU, lumen of the seminiferous tubules; P, pachytene spermatocytes; PR, preleptotene spermatocytes; RS, round spermatids; S, Sertoli cells; SG, spermatogonia. Scale bar: 25 µm in A,B; 55 µm in C.

 


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  		Fig. 3. Distribution of the TIF1ß protein in control (TIF1ßL2/L2:PrP-Cre-ERT(0/0)) and experimental (TIF1ßL2/L2:PrP-Cre-ERT(tg/0)) testes, 1 day (B), 6 weeks (C-F) and 8 weeks (A) after tamoxifen treatment. All the seminiferous tubules displayed here correspond to histologically normal stage VII tubules. C-F are from the same experimental testis. ES, elongated spermatids; P, pachytene spermatocytes; PR, preleptotene spermatocytes; RS, round spermatids; S, Sertoli cells. Immunostaining for TIF1ß (pink and red signals) with DAPI counterstain (blue signal). Scale bar: 100 µm in A,B; 50 µm in C-F.

 


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  		Fig. 4. Percentages of seminiferous tubules exhibiting an abnormal expression pattern of TIF1ß or displaying vacuoles in TIF1ßL2/L2:PrP-Cre-ERT(0/0) (control) and TIF1ßL2/L2:PrP-Cre-ERT(tg/0) (experimental) mice. The percentage of seminiferous tubules cross sections showing abnormal expression of TIF1ß (i.e. partial staining of spermatocyte and/or spermatid layers along the circumference of a given tubule, instead of staining along its whole circumference) was determined by immunohistochemistry 2 weeks after tamoxifen treatment (left). The percentage of degenerating (vacuolated) versus normal tubules was determined 8 weeks after tamoxifen treatment (right). Results are mean±s.e.m. from at least three animals of each genotype and age (***P<0.0001 according to Fischer’s PLSD).

 


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  		Fig. 5. Histological aspect of TIF1ß(L2/L2):PrP-Cre-ERT(0/0) control testis (A,C) and TIF1ßL2/L2:PrP-Cre-ERT(tg/0) experimental testis (B,D-H), 8 weeks after tamoxifen treatment. The seminiferous tubule in C is at stage VII. The abnormal seminiferous tubules in D,G are difficult to stage because of the complete absence of spermatocyte populations; however, they have features in common with a stage VII tubule (‘VII’), namely the aspect of acrosomes in round spermatids (not visible at this magnification) and/or alignment of elongated spermatids. Groat Hematoxylin and Malloy’s trichrome (A-F) or immunolabeling for TIF1ß and DAPI counterstaining (G,H). L, Leydig cells; NT, tubules showing normal germ cell associations; P, pachytene spermatocytes; PR, preleptotene spermatocytes; RS, round spermatids; S, Sertoli cells; ST, Sertoli cell-only seminiferous tubules. The arrowhead in E indicates exfoliated round spermatids. Scale bar: 100 µm (A,B), 15 µm (C,D,F) and 20 µm (E,G,H).

 


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  		Fig. 6. Tunnel labeling (A-D) of the testis and histological detection (E,F) of immature germ cells in the epididymis of TIF1ßL2/L2:PrP-Cre-ERT(0/0) control and TIF1ßL2/L2:PrP-Cre-ERT(tg/0) experimental males 8 weeks after tamoxifen treatment. The green fluorescent signal corresponds to nuclei containing DNA fragments. Arrowheads indicate exfoliated round spermatids. E, epithelium of the cranial portion of the epididymis; ES, elongated spermatids; P, pachytene spermatocytes; RS, round spermatids; S, Sertoli cells; SZ, spermatozoa; T, seminiferous tubules. Scale bar: 100 µm in A,B; 20 µm in C-F.

 

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