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First published online September 17, 2008
doi: 10.1242/10.1242/jcs.035071

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STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation

Manuel Mark^*, Hugues Jacobs, Mustapha Oulad-Abdelghani, Christine Dennefeld, Betty Féret, Nadège Vernet, Carmen-Alina Codreanu, Pierre Chambon and Norbert B. Ghyselinck^*

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Inserm U596, CNRS UMR7104, Illkirch F-67400 France

View larger version (35K): [in this window] [in a new window]   Fig. 1. Targeted disruption of the Stra8 gene. (A) Structure of the targeting vector and partial restriction map of the WT Stra8 locus before [WT (+) allele] and after (L3 allele) homologous recombination, as well as after Cre-mediated excision (L– allele). Black boxes (1-5) indicate exons. The locations of restriction sites (E, EcoRI; N, NcoI; 47, Eco47III, X, XbaI) are indicated. Arrowhead flags represent loxP sites. neo, neomycin-resistance cassette; pBS-SK, pBluescript II SK+. Arrows indicate the location of primers #1, #2 and #3, which were used for genotyping. (B) Long-range PCR analysis of tail genomic DNA from mice with the indicated Stra8 genotype using primers #1 and #2 (upper panel), and #1 and #3 (lower panel). The identities of the different alleles are indicated on the right. Note that a large fragment corresponding to the (+) allele is amplified from DNA of WT mice using primers #1 and #3 (lower panel). The asterisk points to an unspecific product. (C) Schematic representation of the Stra8 transcripts that are synthesised from the WT or the mutant loci (left). Open and black segments in boxes 1-5 indicate non-coding and translated regions of the exons, respectively. The location of the ATG codon is specified for each Stra8 isoform. The size of the fragments amplified from the corresponding cDNA using primers #4 and #5 is also indicated. (Right) Results of RT-PCR assays obtained from P15 WT (+/+) and homozygous mutant (L–/L–) testis RNA using primers #4 and #5 (right panel). The size of the fragments is indicated on the right. Negative and positive controls were obtained using water (H2O) and a STRA8-expressing plasmid (pStra8), respectively. As expected, a shorter fragment corresponding to a truncated mRNA is amplified from the Stra8-null-testis RNA samples. The asterisk points to primer dimers.

View larger version (88K): [in this window] [in a new window]   Fig. 2. The Stra8 mutation is a null mutation. (A-K) Absence of the STRA8 protein in germ cells from male mutants. (A,B) Show immunodetection of STRA8 (red signal) on histological sections of testes at 8 weeks of age, and the DAPI nuclear counterstain (blue). (C,D) Show immunodetection of STRA8 (red signal) on histological sections of testes at P5 and the DAPI nuclear counterstain (blue). (E-J) Show immunodetection of STRA8 (red signal) on nuclear spreads from testes at 8 weeks of age and the DAPI counterstain (blue). Note that: (1) in G,J, the superimposition of the immunohistochemical signals (red false colour) and of the DAPI nuclear stain (blue false colour) were converted to a bright-field image using Photoshop, and (2) in E-J, the three panels in each column represent the same nuclei. (K) Western blot analysis of testis proteins (100 g) from adult mice with the indicated genotypes, using an anti-STRA8 polyclonal antibody (upper panel). Excision of exons 2-4 resulted in a null allele, as confirmed by the absence of any STRA8 in Stra8–/– testes (arrow). The asterisk points to an unspecific signal that was detected both in WT and Stra8–/– extracts. The lower panel, showing detection of actin (ACTA), reveals that similar amounts of protein are loaded on each lane (arrow). (L-O) Absence of oocytes and follicles in ovaries from mutant females at 8 weeks of age. Histological sections were stained with haematoxylin and eosin. The genotypes are as indicated. AF/PF/PRF, antral/primary/primordial follicles; E, ovarian surface epithelium; LY, Leydig cells; PR, preleptotene spermatocytes; ST, ovarian stroma; T, seminiferous tubules. Scale bar: 80 m (A-D); 15 m (E-J); 160 m (L,N); 40 m (M,O).

View larger version (117K): [in this window] [in a new window]   Fig. 3. Spermatogenesis in Stra8–/– mutants can progress to an early pachytene-like stage. Histological sections through the testes of WT and Stra8–/– mutants at the age of 8 weeks are shown. (A,B) Overview of seminiferous-tubule sections stained with haematoxylin and eosin. (C) Percentages of the most advanced spermatocytes detected on haematoxylin- and eosin-stained histological sections of 8-week-old WT and Stra8–/– mutants. PR (preleptotene), L (leptotene), Z (zygotene), P (pachytene), D (diplotene) and M (metaphase I) indicate the most advanced spermatocytes found in a given seminiferous-tubule section, whereas 0 indicates tubule sections containing only Sertoli cells, spermatogonia and eventually abnormal metaphase-like cells (M*, see the main text for further details). Stra8–/– mutants display a relative excess in premeiotic (i.e. preleptotene) and early meiotic (i.e. leptotene) spermatocytes, together with a marked deficit in the more advanced zygotene or pachytene spermatocytes and no diplotene spermatocytes. (D-I) Semi-thin histological sections stained with toluidine blue demonstrate the presence, in Stra8–/– mutants, of spermatocyte nuclei with zygotene (Z) and early-pachytene (eP) nuclear morphologies. The latter represent the most developmentally advanced germ-cell types present in Stra8–/– testes, because nuclei with an XY body (the condensed chromatin structure containing the sex chromosomes) that are characteristic of mid-pachynema (see XY in H) were never seen in Stra8–/– testes. S, Sertoli cells. (J-M) Immunofluorescence staining for SYCP3 and TRIM24. (J,K) The SYCP3 immunolabelling pattern (red), which is characteristic of early-pachytene spermatocytes (eP), is shown. The DAPI nuclear counterstain is blue. R, round spermatids. (L,M) TRIM24 (red), which is not detected in WT maturing spermatocytes prior to mid-pachynema, is undetectable in mutant meiocytes. The DAPI nuclear counterstain is blue. SG, spermatogonia. Roman numerals in D,F,H,L designate stages of the seminiferous-epithelium cycle (Russell et al., 1990). Scale bar: 80 m (A,B); 15 m (D-K); 40 m (L,M).

View larger version (103K): [in this window] [in a new window]   Fig. 4. Stra8–/– preleptotene spermatocytes can achieve a premeiotic S phase and express the meiotic cohesins Rec8 and Smc1b. Histological sections through the testes of WT and Stra8–/– mutants at the age of 8 weeks are shown. (A-G) Photomicrographs illustrating that Stra8 transcripts (purple and red signals) are expressed (1) in all preleptotene spermatocytes and (2) in differentiating A spermatogonia that were identified as A1-A3 on the basis of their presence at precise stages of the seminiferous-epithelium cycle (Chiarini-Garcia and Russell, 2001). (H,J) Immunohistochemical detection of BrdU (red signal) in WT and Stra8–/– preleptotene spermatocytes at 2 hours after its administration. (I,K) Show the same seminiferous tubules as in H,J, but stained with haematoxylin and eosin to allow identification of the cell types on the basis of their nuclear morphology. (L-Q) Immunohistochemical detection of BrdU in seminiferous tubules 8 days after its administration: BrdU that was initially incorporated in preleptotene spermatocytes has been transferred to spermatocytes with pachytene nuclear morphologies both in WT and Stra8–/– males. Note that M,P correspond to superimpositions of fluorescent signals (brown false colour), and of an haematoxylin and eosin stain of the tubule sections displayed in L,O. Also note that N,Q are high-magnification views of the areas delineated by boxes in L,M,O,P. (R-Y) Detection of Rec8 and Smc1b transcripts in the seminiferous epithelium. A1/A2/B, A1/A2/B spermatogonia; P, pachytene spermatocytes; PR, preleptotene spermatocytes; R, round spermatids; S, Sertoli cells; St10/St15/St16, step 10/15/16 spermatids. Roman numerals designate stages of the seminiferous-epithelium cycle. Note that, in C,E,G,S,U,W,Y, the in situ hybridisation signals were converted to a red false colour, and the DAPI nuclear stain was converted to a bright-field image and then to a blue false colour using Photoshop. Scale bar: 80 m (A,H-P); 30 m (B-G,N,Q); 40 m (R-Y).

View larger version (98K): [in this window] [in a new window]   Fig. 5. Abnormal synapsis in Stra8–/– spermatocytes. Immunostaining of surface-spread nuclei from 4-week-old males using antibodies to SYCP1, SYCP3, H2AX and RAD51, as indicated, is shown. L/Z/P, leptotene/zygotene/pachytene spermatocytes; Z/P, designates the most advanced spermatocytes that were present in the mutant testes – these cells shared characters common to zygonema and pachynema. (A-G,P,Q) Formation of the SC is assessed by immunofluorescence on nuclear spreads. The axial elements and the lateral elements of the SC are stained with anti-SYCP3 antibodies (green). The central elements of the SC are stained with anti-SYCP1 antibodies (red). The SC appears yellow from overlapping of SYCP1 and SYCP3 signals. (A) Early leptonema is indistinguishable between WT (not shown) and Stra8–/– spermatocytes. (A-C) Bouquet configurations are unique to Stra8–/– spermatocytes. (D,F) Short fragments of the SC (yellow) have begun to form both in WT and Stra8–/– spermatocytes at early zygonema. (E) In WT pachytene spermatocytes, 19 fully synapsed bivalents are visible along with the XY pair. (G,P,Q) In the Stra8–/– Z/P spermatocytes, the chromosomes are either not synapsed (asterisk) or partially synapsed (white arrows), although fully synapsed chromosome pairs are occasionally seen (arrowheads). In addition, lateral elements are frequently heterosynapsed (blue arrows). (H,J) WT and Stra8–/– spermatocytes express H2AX (red signal) throughout the nucleus during zygonema. (I) In WT spermatocytes during pachynema, H2AX expression becomes restricted to the (almost) unsynapsed XY body. (K) In Stra8–/– spermatocytes at the Z/P stage, H2AX remains widely distributed throughout the nucleus, except in the synapsed regions (arrowed thick green strands), reflecting the near-absolute lack of homologous pairing in these spermatocytes. (L,M) In WT spermatocytes, the number of RAD51 foci (green signal) sharply decreases during the progression from zygonema to pachynema. (N,O) In the most advanced Stra8–/– spermatocytes, the density of RAD51 foci is abnormally elevated but, similar to the WT situation, displays an inverse correlation with the extent of synapsis. Scale bar: 10 m (A-O); 4 m (P,Q).

View larger version (126K): [in this window] [in a new window]   Fig. 6. Stra8–/– aberrant meiocytes, displaying metaphase-like aspects and abnormal spindles, are derived from leptotene spermatocytes. Histological sections at 8 weeks of age are shown. (A,B) Staining with haematoxylin and eosin (A) and toluidine blue (B): the aberrant cells display evenly matched patches of condensed chromatin. (C-J) Immunofluorescence staining (red signal) and DAPI nuclear counterstain (blue) of histological sections for detection of: (C-F) the phosphorylated form of histone H3 (pH3), a marker of highly condensed (i.e. diplotene and metaphase) chromosomes; (G,H) -tubulin, a marker of mitotic spindles; and (I,J) CDK5, a marker of the meiotic spindle. Note that the abnormal metaphase-like chromosomes (in H) are spherically arranged in rosette configurations instead of being neatly aligned at the cell equator (in G). (K) Superimposition of two adjacent sections processed for detection of apoptotic cells (TUNEL assay, green signal) and for detection of pH3 (red signal). The absence of colocalisation of the green and red signals indicates that the Stra8–/– aberrant meiocytes do not undergo apoptosis. (L-Q) Immunohistochemical detection of BrdU (red signals) in seminiferous tubules 24 hours after its administration. (L-O) BrdU that was incorporated into preleptotene spermatocytes (see Fig. 3) has been transferred to leptotene spermatocytes in both WT and Stra8–/– testes. (P,Q) In Stra8–/– testes, BrdU has also been transferred to the aberrant metaphase-like meiocytes during this 24-hour period. M,O,Q correspond to the same tubule sections as in L,N,P, respectively, now stained with haematoxylin and eosin. The insets in M,O,Q correspond to high magnifications of the meiocytes arrowed in L,N,P, respectively. L/D, leptotene/diplotene spermatocytes; M, cells in meiotic metaphase (WT); M*, metaphase-like (Stra8–/–) cells; S, Sertoli cells. Roman numerals designate stages of the seminiferous-epithelium cycle. Scale bar: 40 m (A,C-F,I,J,L-Q); 20 m (B); 10 m (G,H); 160 m (K).

View larger version (108K): [in this window] [in a new window]   Fig. 7. Characterisation of the metaphase-like cells in Stra8–/– testes. (A-H) Immunodetection of SYCP3 (green signal), H2AX (red signal) and DAPI nuclear counterstain (blue) on histological sections. (I-L) Double immunolabelling for detection of SYCP3 (green signal), H2AX (red signal) and DAPI nuclear counterstain (blue) on nuclear spreads. (M-P) Giemsa-stained metaphase spreads. Note that panels M,N correspond to WT meiotic and mitotic (i.e. spermatogonial) metaphases, respectively. L/Z, leptotene/zygotene spermatocytes; M, cells in meiotic metaphase (WT); M*, metaphase-like (Stra8–/–) cells; XII, stage XII of the seminiferous-epithelium cycle. Scale bar: 30 m (A-H); 10 m (I-P).

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