6 research outputs found
RAD51 and DMC1 foci persist in <i>Mcmdc2</i><sup><i>-/-</i></sup> oocytes.
<p>(<b>a, c, e)</b> Immunostaining of SYCP3 along with RAD51 (<b>a</b>), DMC1 (<b>c</b>) or γH2AX (<b>e</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i><sup><i>+/+</i></sup>, or zygotene-pachytene <i>Mcmdc2</i><sup><i>-/-</i></sup> oocytes. Oocytes were collected from the ovaries of littermate fetuses at 18dpc, which is a time point when most wild-type oocytes are in the late pachytene stage. RAD51 and DMC1 foci are largely absent from synapsed chromosomes in <i>Mcmdc2</i><sup><i>+/+</i></sup> oocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i><sup><i>-/-</i></sup> oocytes. (<b>e</b>) γH2AX is largely absent from the synapsed chromosomes of the <i>Mcmdc2</i><sup><i>+/+</i></sup> oocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i><sup><i>-/-</i></sup> oocyte. Scale bars; 10μm. (<b>b, d</b>) Numbers of RAD51 (<b>b</b>) or DMC1 (<b>d</b>) foci are shown in <i>Mcmdc2</i><sup><i>+/+</i></sup> and <i>Mcmdc2</i><sup><i>-/-</i></sup> oocytes at 18dpc. Median numbers of foci are marked, and n corresponds to the number of analyzed oocytes in two pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in <i>Mcmdc2</i><sup><i>-/-</i></sup> than in <i>Mcmdc2</i><sup><i>+/+</i></sup> oocytes (Mann Whitney test).</p
MutSγ and MutLγ foci formation are defective in <i>Mcmdc2</i><sup><i>-/-</i></sup> meiocytes.
<p>(<b>a, b, e, f</b>) Immunostaining of SYCP3 together with MSH4 (<b>a, b</b>) or MLH1 (<b>e, f</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i><sup><i>+/+</i></sup> or zygotene-pachytene <i>Mcmdc2</i><sup><i>-/-</i></sup> meiocytes. (<b>a, b</b>) MSH4 foci are readily detected along synapsed axes of pachytene spermatocytes and oocytes (16dpc). MSH4 foci numbers are much lower in <i>Mcmdc2</i><sup><i>-/-</i></sup> meiocytes. (<b>e, f</b>) Typically, a single MLH1 focus is detected along each synapsed axis pair of <i>Mcmdc2</i><sup><i>+/+</i></sup> pachytene spermatocytes and oocytes (from ovaries of newborn mice). MLH1 foci are not present along the unsynapsed axes of <i>Mcmdc2</i><sup><i>-/-</i></sup> meiocytes. Scale bars; 10μm. (<b>c, d</b>) Numbers of MSH4 foci in <i>Mcmdc2</i><sup><i>+/+</i></sup> and <i>Mcmdc2</i><sup><i>-/-</i></sup> spermatocytes and oocytes. (<b>c</b>) Spermatocytes were examined at leptotene (lepto), early zygotene (e zygo) in <i>Mcmdc2</i><sup><i>+/+</i></sup> and <i>Mcmdc2</i><sup><i>-/-</i></sup>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i><sup><i>+/+</i></sup> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i><sup><i>-/-</i></sup> mice. MSH4 foci numbers are significantly lower in <i>Mcmdc2</i><sup><i>-/-</i></sup> than in <i>Mcmdc2</i><sup><i>+/+</i></sup> spermatocytes from early-zygotene stage onwards (Mann Whitney test). (<b>d</b>) Oocytes with fully formed axes (late zygotene and early pachytene) were examined from fetal ovaries at the 16dpc developmental time point. MSH4 foci numbers are significantly lower in <i>Mcmdc2</i><sup><i>-/-</i></sup> than in <i>Mcmdc2</i><sup><i>+/+</i></sup> oocytes (Mann Whitney test). (<b>c, d</b>) Median numbers of foci are marked, and n corresponds to the number of analyzed meiocytes in two pooled experiments.</p
RAD51 and DMC1 foci persist in <i>Mcmdc2</i><sup><i>-/-</i></sup> <i>s</i>permatocytes.
<p>(<b>a, b, e</b>) Immunostaining showing SYCP3 together with RAD51 (<b>a</b>), DMC1 (<b>b</b>) or γH2AX (<b>e</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i><sup><i>+/+</i></sup>, late zygotene-pachytene <i>Mcmdc2</i><sup><i>-/-</i></sup>, <i>Spo11</i><sup><i>-/-</i></sup>, and <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes. RAD51 and DMC1 foci are present at comparatively high density along the axes of unsynapsed sex chromosomes (<b>a, b</b>, asterisk), and are largely absent from synapsed autosomes of <i>Mcmdc2</i><sup><i>+/+</i></sup> spermatocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i><sup><i>-/-</i></sup> spermatocytes. Absence of RAD51 and DMC1 foci is shown in <i>Spo11</i><sup><i>-/-</i></sup> and <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes. (<b>e</b>) γH2AX preferentially accumulates on the partially synapsed sex chromosomes of the <i>Mcmdc2</i><sup><i>+/+</i></sup> spermatocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i><sup><i>-/-</i></sup> spermatocytes. γH2AX is largely restricted to the sex chromatin in wild-type pachytene spermatocytes, and to pseudo-sex bodies in <i>Spo11</i><sup><i>-/-</i></sup> and <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes. Scale bars; 10μm. (<b>c, d</b>) Numbers of RAD51 (<b>c</b>) or DMC1 (<b>d</b>) foci are shown in leptotene (lepto), early zygotene (e zygo) in <i>Mcmdc2</i><sup><i>+/+</i></sup> and <i>Mcmdc2</i><sup><i>-/-</i></sup>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i><sup><i>+/+</i></sup> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i><sup><i>-/-</i></sup> spermatocytes. Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in three pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in zygotene-pachytene <i>Mcmdc2</i><sup><i>-/-</i></sup> spermatocytes than in late-zygotene or early-mid-pachytene <i>Mcmdc2</i><sup><i>+/+</i></sup> spermatocytes (Mann Whitney test).</p
MCMDC2 is not required for extensive non-homologous synaptonemal complex formation in the <i>Spo11</i><sup><i>-/-</i></sup> background.
<p>(<b>a</b>) SYCP3 (axis marker) and SYCP1 (synaptonemal complex marker) were detected by immunofluorescence on nuclear surface spreads of zygotene-pachytene <i>Mcmdc2</i><sup><i>-/-</i></sup>, <i>Spo11</i><sup><i>-/-</i></sup> or <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes. Whereas comparatively few synaptonemal complex stretches are detected in the <i>Mcmdc2</i><sup><i>-/-</i></sup>spermatocyte, extensive non-homologous synaptonemal complex formation is seen in the <i>Spo11</i><sup><i>-/-</i></sup> or <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes. Scale bars; 10μm (<b>b</b>) Quantification of SYCP1 stretch numbers in zygotene-pachytene spermatocytes with fully condensed chromosome axes of the indicated genotypes. The numbers of synaptonemal complex stretches is significantly higher in <i>Spo11</i><sup><i>-/-</i></sup> or <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes than in <i>Mcmdc2</i><sup><i>-/-</i></sup> (Mann Whitney test). The numbers of synaptonemal complex stretches are not significantly different in <i>Spo11</i><sup><i>-/-</i></sup> or <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/—</i></sup>spermatocytes (p = 0.8639, Mann Whitney test). Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in two (<i>Spo11</i><sup><i>-/-</i></sup> or <i>Spo11</i><sup><i>-/-</i></sup> <i>Mcmdc2</i><sup><i>-/-</i></sup>) or three (<i>Mcmdc2</i><sup><i>-/-</i></sup>) pooled experiments.</p
<i>Mcmdc2</i><sup><i>-/-</i></sup> mice are deficient in germ cells from late meiotic prophase onwards in both sexes.
<p>(<b>a, b</b>) Growth curves of five (<b>a</b>) or three (<b>b</b>) independent lines of <i>Mcmdc2</i><sup><i>+/</i>+</sup> (+/+) and <i>Mcmdc2</i><sup><i>-/-</i></sup> mouse embryonic fibroblasts. Cells were grown either without aphidicolin treatment (<b>a</b>) or with aphidicolin treatment for the first 24 hours (<b>b</b>), where 1μM aphidicolin was added at day 0. (<b>a, b</b>) Cell numbers were determined at the indicated time points in three technical replicates of each fibroblast line. Means and standard deviations of the medians of technical triplicates are shown. Growth curves of <i>Mcmdc2</i><sup><i>+/</i>+</sup> and <i>Mcmdc2</i><sup><i>-/-</i></sup> mouse embryonic fibroblasts are not significantly different (<b>a</b>: p = 0.8201, <b>b</b>: p = 0.9932, two-way ANOVA test). (<b>c</b>) Images of <i>Mcmdc2</i><sup><i>+/</i>+</sup> (+/+) and <i>Mcmdc2</i><sup><i>-/-</i></sup> (-/-) testes (upper panel) and ovaries (lower panel). Scale bars; 500μm. (<b>d</b>) Cryosections of testes from adult <i>Mcmdc2</i><sup><i>+/+</i></sup> and <i>Mcmdc2</i><sup><i>-/-</i></sup> mice. DNA was detected by DAPI, histone H1T (marker of spermatocytes after mid-pachytene) and nuclear cleaved PARP1 (marker of apoptotic cells) were detected by immunostaining. Outlines of testis tubules are marked by dashed lines. The upper panels of <b>d</b> show stage V-VI and VII-VIII wild-type testis tubules, which contain several layers of germ cells at distinct spermatogenic stages: Sertoli cells (Se), spermatogonia B (SgB, stage V-VI), preleptotene (pl, stage VII-VIII), mid-pachytene (pa, stage V-VI), late-pachytene (pa, stage VII-VIII) spermatocytes, post-meiotic spermatids (sd) and spermatozoa (sp). Lower panels of <b>d</b> show that <i>Mcmdc2</i><sup><i>-/-</i></sup> meiocytes underwent apoptosis at a stage corresponding to wild-type mid-pachytene in stage IV tubules. Consequently, spermatocytes were not found in the inner layers of testis tubules beyond stage IV, and post-meiotic spermatids and spermatozoa were also missing from <i>Mcmdc2</i><sup><i>-/-</i></sup> testes. To illustrate this, stage IV, V-VI and VII-VIII tubules of <i>Mcmdc2</i><sup><i>-/-</i></sup> mice are shown. Apoptotic (ap) and non-apoptotic early-mid pachytene (pa) spermatocytes are shown in the stage IV tubule, which was identified by the presence of mitotic intermediate spermatogonia (m) and intermediate spermatogonia (Int). Stage V-VI and VII-VIII tubules contain somatic Sertoli cells (Se) and spermatogonia B (SgB) or preleptotene (pl) spermatocytes, respectively, but more advanced spermatogenic cells are missing. Due to elimination at mid-pachytene, histone H1T positive cells are missing from <i>Mcmdc2</i><sup><i>-/-</i></sup> testis tubules. (<b>e</b>) NOBOX (oocyte marker) was detected by immunofluorescence on cryosections of ovaries from 6-week-old mice. DNA was stained by DAPI. Oocytes in primordial (pd) and secondary (s) follicles are shown in the section of a wild-type ovary. In contrast, oocytes are not detected in the shown <i>Mcmdc2</i><sup><i>-/-</i></sup> ovary section. (<b>d, e</b>) Scale bars; 50μm.</p
Preferential expression of <i>Mcmdc2</i> in the gonads, and <i>Mcmdc2</i> targeting in mice.
<p>(<b>a</b>) Expression of <i>Mcmdc2</i> and a “house-keeping” gene (<i>S9</i>) in testis and a somatic tissue mix measured by RT-PCR. cDNAs were prepared from four RNA mixtures: (1) Equal amounts of RNAs from 17 somatic tissues (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006393#sec012" target="_blank">Materials and Methods</a> for the tissue list) were mixed and 1μg of the resulting mixture was used for RT (17 somatic tissues). (2) Mixture “1” supplemented with testis RNA at a concentration equal to that of the individual somatic RNAs (17 somatic tissues + 1x testis). (3) Mixture “1” supplemented with testis RNA at a concentration equal to five times that of the individual somatic RNAs (17 somatic tissues + 5 x testis) (4) Mixture “3” with no RT (17 somatic tissues + 5xtestis noRT). <i>Mcmdc2</i>-specific PCR-products were amplified preferentially from templates that contained testis cDNA. (<b>b</b>) <i>Mcmdc2</i> targeting strategy. Schematics of the targeting construct, the wild-type (WT) and the modified <i>Mcmdc2</i> genomic locus. Black boxes represent exons (not to scale). Recombination at the homology arms (HA) of the targeting construct modifies intron 4 by introducing: 1) an additional exon (SA-IRES-LacZ) that contains a strong splice acceptor site (SA) and poly-adenylation site (left grey box), 2) a transcriptional unit that contains the strong housekeeping human ß-Actin promotor <i>(hBactP)</i> driving the neomycin (Neo) resistance gene as a selection marker. This modification of intron 4 also disrupts the <i>Mcmdc2</i> open reading frame after the 95th codon <i>(Mcmdc2</i><sup><i>insertion</i></sup> allele). Recombination catalyzed by FLPe at FRT sites removes the SA-IRES-LacZ exon and the hBactP-Neo gene, and restores the MCMDC2 ORF (<i>Mcmdc2</i><sup><i>restored</i></sup>). <i>Mcmdc2</i><sup><i>restored</i></sup> is a functional allele that can be disrupted by Cre-mediated recombination between loxP sites (<i>Mcmdc2</i><sup><i>deletion</i></sup>). Excision of exon 5–7 causes a frameshift after the 80th codon. Cre-mediated recombination between loxP sites of a <i>Mcmdc2</i><sup><i>insertion</i></sup> allele results in <i>Mcmdc2</i><sup><i>insertion-deletion</i></sup> allele. The positions of PCR-genotyping primers are indicated. Red bars mark the 3`and the internal Southern blot probes; the predicted length of restriction fragments is indicated. (<b>c</b>) Southern blot of DNA from wild-type (+/+) and targeted <i>Mcmdc2</i><sup><i>+/insertion</i></sup> (<i>+/i</i>) embryonic stem cell clones (C6 and F7) that were used to derive two independent mouse lines. DNA was digested with Eco31I and hybridized with an internal probe for LacZ (left panel), or DNA was digested with BclI and hybridized with a 3’ probe (right panel). The blots indicate a single integration of the targeting cassette in the <i>Mcmdc2</i> locus. (<b>d</b>) RT-PCR was used to detect <i>Mcmdc2</i> and "house-keeping" <i>Rps9 (S9)</i> transcripts in testes of wild-type and <i>Mcmdc2</i><sup><i>-/-</i></sup> (insertion-deletion) mice. Oligo-pairs specific to <i>Mcmdc2</i> exon 3 and 4, 5 and 6, 6 and 7, 8 and 9, or 10 and 11 were used.</p