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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00975

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Disruption of spermatogenesis in mice lacking A-type lamins

Manfred Alsheimer^1,*, Bodo Liebe^2,*, Lori Sewell³, Colin L. Stewart³, Harry Scherthan^2, and Ricardo Benavente^1,

¹ Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
² Max-Planck-Institute for Molecular Genetics, 14195 Berlin, Germany
³ Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702-1201, USA

View larger version (131K): [in a new window]   Fig. 1. Light microscopy of testis sections from 31-day-old wild-type (A), heterozygous (B) and Lmna–/– (C) littermates. Sections were stained with Hematoxylin. In the corresponding insets, the whole testes are also shown for comparison. (D-F) Apoptotic cells are revealed by TUNEL, which was performed on sections of testes from 31-day-old wild-type, heterozygous and Lmna–/– littermates, respectively. These sections are counterstained with Methyl Green. Scale bars: 100 µm (2 mm in the insets).

View larger version (100K): [in a new window]   Fig. 2. Light microscopy of epididymis sections from adult wild-type (A) and Lmna–/– (B) littermates reveals that the epididymis of the knockout is fluid filled and does not contain sperm. Sections are stained with Hematoxylin and Eosin. Scale bars: 50 µm.

View larger version (11K): [in a new window]   Fig. 3. Frequencies of meiotic stages in wild-type, heterozygous and Lmna–/– testes suspensions. Mid-preleptotene and bouquet frequencies are based on Maj.Sat/T2AG3 FISH (Scherthan et al., 1996) analysis of >1950 meiocytes/genotype. H1t+, histone H1t-expressing spermatocytes as identified by anti-H1t immunofluorescence (Scherthan et al., 2000a). All other stages were determined by SCP3 immunofluorescence analysis of at least 200 spermatocytes of each genotype. The absence of A-type lamins induces a highly significant increase in early prophase stages, while later stages (H1t+ late pachytene and diplotene) are significantly reduced, even in the heterozygote. *Highly significantly different (2, P<0.001) and #significantly different (2, P<0.01) compared with WT. aMid-preleptotene and bouquet values determined by Cen/Tel FISH (Scherthan et al. 2000a).

View larger version (178K): [in a new window]   Fig. 4. Cross sections of testes from adult wild-type (A,D) and Lmna–/– (B,C,E,F) mice. Sections are stained with Hematoxylin (A,B,D,E) or in situ labeled using the TUNEL assay and counterstained with Methyl Green (C,F). (A-C) Stage V, (D-F) stage VII in the epithelium cycle. (G) Transmission electron microscopy of a seminiferous tubule from a 22-day-old Lmna–/– mouse. S, Sertoli cell, P, pachytene spermatocyte. Arrows in E and arrowheads in G denote some of the dead spermatocytes. (F) Higher magnification of a dead spermatocyte. N, nucleus, C, cytoplasm. Arrows denote SC structures. Scale bars: 20 µm (A-G), 0.5 µm (H).

View larger version (100K): [in a new window]   Fig. 5. Electron microscopy showing the attachment site of the axial elements (AEs) of the synaptonemal complex (SC) of autosomal bivalents to the nuclear envelope (NE) in 22-day-old wild-type (A) and Lmna–/– (B) littermates. Both telomeric attachments are structurally undistinguishable. Scale bars: 0.2 µm.

View larger version (54K): [in a new window]   Fig. 6. Spread spermatocytes of 28-day-old Lmna–/– (A-C) and heterozygous (D) littermates stained for the axial element protein SCP3 (green, FITC) and the telomere protein Trf1 (red, rhodamine). Complete synaptonemal complexes (SCs; thick green rods) display Trf1 signals (red dots) at their termini as do the numerous long unpaired axial elements. (A) Zygotene-like Lmna–/– spermatocyte with numerous unpaired axes. (B) Zygotene-like Lmna–/– spermatocyte with a short univalent and numerous unpaired axes. (C) Lmna–/– spermatocyte at pachytene exhibits univalent X (arrow) and Y chromosomes (long arrow). (D) Pachytene spermatocyte of a heterozygous control littermate with normal SCs and a sex chromosome bivalent (arrowhead) with Trf1 signals at all telomeres. Scale bars: 10 µm.

View larger version (103K): [in a new window]   Fig. 7. Light microscopy of Toluidine Blue-stained ovary sections from 28-day-old heterozygous (A) and Lmna–/– (B) littermates. In agreement with female fertility, no overt differences are apparent between the genotypes. Scale bars: 50 µm.