Volume 5, Issue 1, Pages 73-83 (January 2000) The Murine SCP3 Gene Is Required for Synaptonemal Complex Assembly, Chromosome Synapsis, and Male Fertility  Li Yuan, Jian-Guo Liu, Jian Zhao, Eva Brundell, Bertil Daneholt, Christer Höög  Molecular Cell  Volume 5, Issue 1, Pages 73-83 (January 2000) DOI: 10.1016/S1097-2765(00)80404-9

Figure 1 Generation of SCP3-Deficient Mice (A) Gene targeting strategy. The restriction maps of the wild-type gene, the mutant SCP3 gene, and the knockout construct are shown. The numbered boxes (I–VI) denote the exons, the first in-frame ATG codon being located in exon I. The neo marker replaces exons I–III in the mutant allele. The Pst1 fragments that are detected by a 5′ probe in Southern blotting experiments are 12.5 kb in the wild type and 5.9 kb in the mutant, respectively. P, Pst1; B, BglII; St, StuI; Sn, SnaBI. (B) Southern blot analysis of representative offsprings from heterozygous matings. +/+, wild type; +/−, heterozygous; +/+, knockout animal. The DNA was digested with PstI and hybridized with a 5′ probe. The 12.5 kb bands are faint due to inefficient blotting of DNA. (C) Northern blot analysis. Total testicular RNA (10 μg) was hybridized with the full-length SCP3 cDNA probe or with a mouse β-actin probe as a control. The SCP3 probe detects the expected 1 kb RNA band, shown previously to correspond to SCP3 RNA (Lammers et al. 1994). (D) Western blot analysis of protein extracts from testis using an affinity-purified anti-SCP3 serum. The detected SCP3 protein migrates as a 30 kDa protein as previously shown (Liu et al. 1996). The anti-lamin antiserum was used to monitor the amount of proteins loaded in each well. Equal amounts of proteins were loaded; however, due to the absence of haploid cells in the testis of the −/− animals, more of the lamin protein is seen in these protein extracts. Molecular Cell 2000 5, 73-83DOI: (10.1016/S1097-2765(00)80404-9)

Figure 2 Spermatogenesis Is Disrupted in SCP3−/− Mice Anatomical and morphological analysis of testis from wild-type and mutant animals. Gross appearance of testes from representative animals with different genotypes (A–C), wild-type (+/+), heterozygous (+/−), and knockouts (−/−). The testes from the SCP3-deficient mouse are clearly smaller than the testes from wild-type and heterozygous animals. Hematoxylin and eosin stained cross sections of testis from adult (D and E) and juvenile (F–L) mice. A full complement of cells was observed in wild-type testis (D) (SP, spermatogonia; LZ, leptotene/zygotene spermatocytes; PS, pachytene spermatocytes; PD, pachytene/diplotene spermatocytes; RS, round spermatids; ES, elongated spermatids). Note the vacuolar structures, the multiple layers of spermatocytes, and the absence of postmeiotic cells in the −/− adult animals (E). The juvenile mice were 10 days of age (F and G), 12 days of age (H and I), and 14 days of age (J–L) Arrow points to an example of an abnormal germ cell (I). Scale bars, 0.2 cm (A–C), 1 μm (D–L). Molecular Cell 2000 5, 73-83DOI: (10.1016/S1097-2765(00)80404-9)

Figure 3 The Spermatocytes in the Testis of SCP3−/− Mice Undergo Apoptosis In situ labeling of apoptotic cells in sections of the seminiferous tubules from wild-type (+/+) and mutant (−/−) animals using the TUNEL assay. Juvenile animals 10 days of age (A and B), 12 days of age (C and D), or adult animals were analyzed (E and F). Note the occurrence of apoptotic cells (dark brown) in the seminiferous tubules of the SCP3−/− animals (D and F). Three distinct types of tubules (labeled I–III) were observed in both adult and juvenile testis. Type II tubules were labeled by the TUNEL assay. Scale bars, 1 μm. Molecular Cell 2000 5, 73-83DOI: (10.1016/S1097-2765(00)80404-9)

Figure 6 The Expression of Rad51, SCP1, and RPA in SCP3−/− Spermatocytes Spermatocytes prepared from wild-type (A, C, and E–G) and knockout animals (B, D, and H–J) were fixed and analyzed by immunofluorescence microscopy. The distribution of Rad51 (red) suggests that the Rad51 foci form arrays in both wild-type (A) and mutant cells (B). The arrows in (A) and (B) point to arrays of Rad51 foci. DNA was labeled by DAPI (blue). SCP1 and CREST antisera were used to label the SC (green) and the centromeres (red) in wild-type (C) and mutant (D) spermatocytes, respectively. The fibers in the SCP3−/− spermatocytes (D) were shorter and not associated with the centromeres (labeled by CREST in red/yellow and indicated by arrows). The SCP1 fibers in the mutant cells also display axial interruptions (arrowheads in [D]). DNA was labeled by DAPI (blue). The distribution of SCP1 and RPA in wild-type (E–G) and mutant (H–I) spermatocytes suggest that the two proteins colocalize. SCP1 (E and H), RPA (F and I), and merged images (G and J). Scale bars, 1 μm (A–D), 2 μm (E–J). Molecular Cell 2000 5, 73-83DOI: (10.1016/S1097-2765(00)80404-9)

Figure 4 Homologous Chromosome Pairing Is Defective in SCP3−/− Male Meiotic Cells Spermatocytes prepared from wild-type (A, B, D, and E) and knockout animals (C and F) were fixed and analyzed by immunofluorescence microscopy. Affinity-purified SCP3 and CREST antisera were used to label the SC (green) and the centromeres (red), respectively, in the spermatocytes. The SCP3 antiserum labels the SCs in spermatocytes isolated from wild-type testis (A and B) but not the corresponding cells isolated from SCP3-deficient mice (C). Note that the CREST sera label the same number of centromeres in SCP3-deficient spermatocytes (C) as in wild-type zygotene spermatocytes (A), suggesting that homologous chromosome pairing in SCP3−/− meiotic cells is blocked. To visualize the localization of chromatin, the same cells were also stained with DAPI and CREST (D–F). Scale bar, 1 μm. Molecular Cell 2000 5, 73-83DOI: (10.1016/S1097-2765(00)80404-9)

Figure 5 The Axial Elements and the SC Fail to Form in SCP3−/− Spermatocytes Meiotic chromosomes were spread, stained using a silver nitrate staining method that labels the LEs and the SCs, and analyzed using light microscopy (A and B) or electron microscopy methods (C–E). Examples of distinctly stained SCs in wild-type spermatocytes are shown ([A], indicated by arrows), whereas cells from SCP3−/− testis having a nuclear size and morphology corresponding to spermatocytes failed to display SCs ([B], indicated by arrows). At higher magnification, both unpaired and paired axial elements are clearly stained in wild-type spermatocytes in zygotene (C) and pachytene (D), but no such structures are observed in SCP3-deficient spermatocytes (E). Scale bars, 5 μm (A and B), 1 μm (C–E). Molecular Cell 2000 5, 73-83DOI: (10.1016/S1097-2765(00)80404-9)