Volume 7, Issue 3, Pages 509-515 (March 2001) Late Arrest of Spermiogenesis and Germ Cell Apoptosis in Mice Lacking the TBP-like TLF/TRF2 Gene  Igor Martianov, Gian-Maria Fimia, Andrée Dierich, Martti Parvinen, Paolo Sassone-Corsi, Irwin Davidson  Molecular Cell  Volume 7, Issue 3, Pages 509-515 (March 2001) DOI: 10.1016/S1097-2765(01)00198-8

Figure 1 Dynamic Expression of TLF in Developing Male Germ Cells (A) Northern blot analysis of mTLF expression. Total RNA from the tissues shown above each lane was hybridized with a 32P-labeled mTLF cDNA probe. The sizes of the hybridizing mRNAs in nucleotides (nt) are indicated. (B) shows in situ hybridization. Sections of testis from wild-type mice were hybridized with the TLF cDNA anti-sense probe. Dark-field and phase contrast views of a representative section are shown. (C) Sections of the testis from wild-type mice were incubated with the anti-TLF antibody. No significant signal was seen with this antibody using sections from testis of TLF−/− mice, showing that the signal was specific for TLF (data available upon request). The upper panel shows a 20× magnification, and the lower panel shows a 40× magnification. The Hoechst-stained DNA and merged images are presented for each section. The approximate stages of each tubule are indicated. ES, elongating spermatids; PS, late pachytene/diakinetic spermatocytes; RS, round spermatids; MS, maturation phase spermatids; Z, zygotene spermatocytes Molecular Cell 2001 7, 509-515DOI: (10.1016/S1097-2765(01)00198-8)

Figure 2 Inactivation of the Mouse TLF Gene by Homologous Recombination (A) The intron/exon structure of the mTLF gene is schematized. The location of the start codon ATG in exon II and the stop codon TAA in exon VII is indicated along with the diagnostic BglII restriction enzyme sites and the 5′ probe used to detect homologous recombination events. The structure of the disrupted allele is also schematized. “L” indicates the presence of loxP sites flanking the hygromycin resistance cassette. The sizes of the BglII restriction fragments from the wild-type and mutated alleles are indicated. (B) A comparison of the amino acid sequences of mouse TBP and TLF is shown. Identical amino acids are indicated in yellow on a black background, and similar amino acids are boxed in blue. Amino acids were classified as follows: small residues, P, A, G, S, T; hydrophobic, L, I, V, A, F, M, C, Y, W; polar/acidic, D, E, Q, N; and basic, R, K, H. The point of insertion of the hygromycin cassette and the exon III–IV boundary are indicated. (C) A representative Southern blot of mouse tail DNA digested with BglII and hybridized with the 5′ probe. The hybridizing fragments corresponding to the wild-type and disrupted alleles are indicated. The genotype of the mice is indicated above each lane. (D) Total RNA from the testis and brain of a TLF−/− mouse or a wild-type littermate, as indicated above each lane, was hybridized with the 32P-labeled mTLF cDNA probe. The results of hybridizing the same RNA with a control 32P-labeled β-actin probe are shown in the lower panel. (E) Immunoblot of extracts from wild-type and mutant mice, as indicated above each lane, using the anti-TLF antibody shows that TLF is absent from the testis of TLF−/− mice Molecular Cell 2001 7, 509-515DOI: (10.1016/S1097-2765(01)00198-8)

Figure 3 Histological Analysis of the Testis of TLF−/− Mice (A) Comparison of the testes (left panel) and dissected seminiferous tubuli (right panel) from TLF−/− mice and a wild-type littermate. (B) Histological analysis of sections from TLF−/− and wild-type testes. The upper panels show 20× magnification, and the lower panels show 40× and 100× magnifications. The arrows indicate apoptotic (Ap and APO-RS) round spermatids. (C) Histological analysis of epididymides from wild-type and mutant mice. Sp, mature spermatozoa in the lumen of wild-type mice Molecular Cell 2001 7, 509-515DOI: (10.1016/S1097-2765(01)00198-8)

Figure 4 Germ Cell Apoptosis in TLF−/− Mice (A) Apoptotic cells were detected by in situ TUNEL immunofluoresence in testis sections from wild-type and mutant mice, as indicated above each panel. Representative apoptotic cells are indicated. A 20× magnification is shown. (B) Graphical representation of the mean number of apoptotic cells detected in 35 randomly sectioned tubuli from wild-type and mutant mice Molecular Cell 2001 7, 509-515DOI: (10.1016/S1097-2765(01)00198-8)

Figure 5 Analysis of Testis Gene Expression in TLF Mutant Mice (A) Northern blot analysis. The RNA from the mice whose genotype is shown above each lane was hybridized with a 32P-labeled probe to detect the expression of the gene shown below each panel. The TLF gene, whose expression is abolished in mutant animals, is included as a control for the RNA samples. Spam, sperm adhesion molecule 1. (B) shows RT–PCR analysis. Exon-specific primers for the genes indicated below each panel were used in RT–PCR reactions with the RNA from mice whose genotype is indicated above each lane. −RTase shows a control reaction using the RNA from TLF−/− mice without addition of reverse transcriptase. Exon-specific amplifications of the TLF gene were performed as control. MCS, mitochondrial capsule selenoprotein; RT7/ODF, outer dense fiber protein. (C) shows immunoblot analysis. Protein extracts from the testis of mice whose genotype is shown above each lane were probed with antibodies against the proteins shown below each panel. The different isoforms of CREM are indicated Molecular Cell 2001 7, 509-515DOI: (10.1016/S1097-2765(01)00198-8)