PMC

Fig. 2. TLS deficiency is associated with defective spermatogenesis. (A) Photomicrographs of representative sections of mouse testes with the TLS genotype indicated. The arrows in panels 4 and 5 point to degenerating pre-meiotic spermatocytes, and in panels 6 and 7 arrows point to round spermatids that are conspicuously larger in the mutant testes. The asterisks in panel 7 point to deformed elongated spermatids. (B) Photomicrographs of representative sections of newborn mouse testes with the TLS genotype indicated.

Fig. 6. TLS contributes to the in vitro pairing–promoting activity present in nuclei of testicular cells. (A) Proteins extracted from nuclei of testicular cells with the TLS genotypes indicated were resolved by SDS–PAGE, blotted onto nitrocellulose and the blot was subjected to a pairing on membrane assay (POM assay). (B) TLS Western blot performed on a parallel sample. (C) Coomassie staining of a parallel-run gel to control for equal loading of nuclear proteins. Note the absence of the major POMp75 species in the extracts from TLS^–/– testes.

Fig. 1. Normal androgen action and internal genitalia of TLS^–/– mice. (A) TLS, EWS and TAF_II68 Western blots of whole-cell extracts from mouse embryonic fibroblasts with the TLS genotypes indicated. (B) Photomicrograph of the internal genitalia of adult (9–week-old) wild-type and TLS^–/– male sibling mice. The bar corresponds to 5 mm. (C) Northern blot of kidney mRNA from female and castrated male mice injected with testosterone (4 μg/g/day) and hybridized with the androgen-responsive SA gene (upper panel) and β–tubulin (lower panel). Note the normal androgen responses of the mutant mice.

Fig. 7. Testicular TLS associates with RNA. (A) In vivo UV cross-linking of RNA species to TLS in testicular cells. Suspensions of freshly isolated testicular cells with the indicated TLS genotypes were irradiated with UV and the cellular TLS was solubilized by RNase A or DNase I treatment as indicated and immunoprecipitated with anti-TLS or control (anti-CHOP) antibodies. Polynucleotides in the immunoprecipitates were end-labeled with ³²P and the TLS-labeled polynucleotide complex was resolved by 10% SDS–PAGE and subjected to autoradiography. (B) The labeled material in (A) was excised from the gel, digested with proteinase K, recovered by ethanol precipitation, divided into three aliquots that were subjected to repeat digestion with RNase A or DNase I and resolved on a 20% acrylamide–8 M urea gel. Note that DNase I digestion fails to degrade the labeled species whereas RNase A degrades it.

Fig. 5. Increased sensitivity of TLS^–/– cells and animals to ionizing irradiation. (A) Day 7 counts of MEFs with the TLS genotypes indicated. The cells were irradiated at day zero with the indicated dose of γ–rays. The number of cells on the plate at day 7 is expressed as a fraction of the cell count on an identical plate at 24 h after plating. Shown are means and SEM of a representative experiment, performed in triplicate and reproduced three times using different pools of MEFs. (B) Survival curve of a cohort of mice discordant for TLS genotype that had received 700 cGR of γ–rays on day 1. Each of the 20 TLS^–/– mice was matched with one or more siblings of TLS^+/+ or TLS^+/– genotype.

Fig. 3. TLS immunostaining and meiotic progression in wild-type and mutant testes. (A) Formalin-fixed and paraffin-embedded 5 μm sections of wild-type (panels 1 and 3) and mutant testes (panel 2) were reacted with the 4H11 anti-TLS monoclonal antibody, a horseradish peroxidase-conjugated secondary antibody, and the stain developed with diaminobenzidine and counterstained with hematoxylin. Specific cell types in the seminiferous epithelium are identified in panel 3: arrow, pachytene spermatocyte; single arrowhead, round spermatid; double arrowhead, Sertoli cell. (B) Chromosomal spreads of a pachytene/early diplotene spermatocyte fixed and stained for a marker of the axial elements (SCP3), TLS or a chromatin-binding dye (DAPI). The arrowhead points to the sex body that contains the partially synapsed X–Y chromosome pair and is a region of the nucleus with very little TLS staining.

Fig. 4. Analysis of synaptonemal complex formation in TLS^–/– spermatocytes by immunolocalization of SCP3 (white) and RAD51 (green) in chromosomal spreads. Panel 1: normal zygotene nucleus from wild-type mouse. Note that synapsis is occurring (red arrows) even as axial elements are still forming. RAD51 localizes to unsynapsed axes and begins to disappear once synapsis has occurred. Panel 2: zygotene nucleus exhibiting synaptic defects. Note the long axial elements that have almost completely formed with little synaptic activity. Some synapsis appears to be non-homologous, based on the differences in length of axial elements (red arrow). Panel 3: zygotene nucleus with major synaptic problems. Synapsed axes show a loss of RAD51 (red arrowhead), while the non-homologously synapsed axes (red arrow) and unsynapsed axes (white arrow) are coated with RAD51. Panel 4: pachytene nucleus with unsynapsed axes and non-homologous synapsis. Considering that the non-homologously synapsed axes involve more than one SC (red arrows), there are >20 axes present in this nucleus. The single axes with a RAD51 coating (white arrows) did not synapse with their homologs. Panel 5: pachytene nucleus containing non-homologously synapsed axes (red arrow). The non-homologously synapsed axes are emphasized by the large amount of RAD51. Also note the abnormal RAD51 bridge connecting two parts of one SC (white arrowhead). Panel 6: high magnification views of abnormal SCs from mutant pachytene nuclei. Incomplete synapsis of two SCs is evident from the double rows of RAD51 along the SCs' lengths (small white arrows) (i and ii). SCP3 staining of these SCs shows that they are thicker than normal, a characteristic of axes that are not fully synapsed. Several axes with partial non-homologous synapses are shown (iii). An abnormal RAD51 bridge has formed between non-homologous regions of the same bivalent (white arrowhead) (iv). SCs containing irregularly shaped RAD51 foci that ‘hang off’ the edges of the SCs, a configuration not observed in normal spermatocytes, are shown (small red arrows) (v).