Analysis of gene expression data from Massive Parallel Sequencing identifies so far uncharacterised regulators for meiosis with one candidate being fundamental for prophase I in male and female meiosis

Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-­‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search. Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knock­out oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search

Analysis of gene expression data from Massive Parallel Sequencing identifies so far uncharacterised regulators for meiosis with one candidate being fundamental for prophase I in male and female meiosis

Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-­‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search. Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knock­out oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search

Analysis of gene expression data from Massive Parallel Sequencing identifies so far uncharacterised regulators for meiosis with one candidate being fundamental for prophase I in male and female meiosis

Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-­‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search. Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knock­out oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search

Microvascular Architecture of Mouse Urinary Bladder Described With Vascular Corrosion Casting, Light Microscopy, SEM, and TEM

The urinary bladder is a unique organ in that its normal function is storage and release of urine, and vasculature in its wall exhibits specialized features designed to accommodate changes in pressure with emptying and filling. Although we have previously described the fine details of the microvasculature of the urinary bladder of the rabbit and dog, information on the fine details of the microvasculature of the mouse bladder were deemed to be of value because of the increasing use of this species in developing genetic models for studying human disorders. The present study shows that many of the special features of the microvasculature of the mouse urinary bladder are similar to those described in the rabbit and dog, including vessel coiling, abundant collateral circulation, arterial sphincters, and a dense mucosal capillary plexus

Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein.

Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during meiotic recombination

MCMDC2 is not required for extensive non-homologous synaptonemal complex formation in the <i>Spo11</i>^<i>-/-</i> 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>^<i>-/-</i>, <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Whereas comparatively few synaptonemal complex stretches are detected in the <i>Mcmdc2</i>^<i>-/-</i>spermatocyte, extensive non-homologous synaptonemal complex formation is seen in the <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>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>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes than in <i>Mcmdc2</i>^<i>-/-</i> (Mann Whitney test). The numbers of synaptonemal complex stretches are not significantly different in <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>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>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/-</i>) or three (<i>Mcmdc2</i>^<i>-/-</i>) pooled experiments.</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>^{<i>insertion</i>} 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>^{<i>restored</i>}). <i>Mcmdc2</i>^{<i>restored</i>} is a functional allele that can be disrupted by Cre-mediated recombination between loxP sites (<i>Mcmdc2</i>^{<i>deletion</i>}). Excision of exon 5–7 causes a frameshift after the 80th codon. Cre-mediated recombination between loxP sites of a <i>Mcmdc2</i>^{<i>insertion</i>} allele results in <i>Mcmdc2</i>^{<i>insertion-deletion</i>} 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>^{<i>+/insertion</i>} (<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>^<i>-/-</i> (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

RAD51 and DMC1 foci persist in <i>Mcmdc2</i>^<i>-/-</i> <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>^<i>+/+</i>, late zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i>, <i>Spo11</i>^<i>-/-</i>, and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>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>^<i>+/+</i> spermatocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. Absence of RAD51 and DMC1 foci is shown in <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. (<b>e</b>) γH2AX preferentially accumulates on the partially synapsed sex chromosomes of the <i>Mcmdc2</i>^<i>+/+</i> spermatocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. γH2AX is largely restricted to the sex chromatin in wild-type pachytene spermatocytes, and to pseudo-sex bodies in <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>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>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i>^<i>+/+</i> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i>^<i>-/-</i> 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>^<i>-/-</i> spermatocytes than in late-zygotene or early-mid-pachytene <i>Mcmdc2</i>^<i>+/+</i> spermatocytes (Mann Whitney test).</p

MutSγ and MutLγ foci formation are defective in <i>Mcmdc2</i>^<i>-/-</i> 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>^<i>+/+</i> or zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> 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>^<i>-/-</i> meiocytes. (<b>e, f</b>) Typically, a single MLH1 focus is detected along each synapsed axis pair of <i>Mcmdc2</i>^<i>+/+</i> pachytene spermatocytes and oocytes (from ovaries of newborn mice). MLH1 foci are not present along the unsynapsed axes of <i>Mcmdc2</i>^<i>-/-</i> meiocytes. Scale bars; 10μm. (<b>c, d</b>) Numbers of MSH4 foci in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i> spermatocytes and oocytes. (<b>c</b>) Spermatocytes were examined at leptotene (lepto), early zygotene (e zygo) in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i>^<i>+/+</i> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i>^<i>-/-</i> mice. MSH4 foci numbers are significantly lower in <i>Mcmdc2</i>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> 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>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> 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>^<i>-/-</i> 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>^<i>+/+</i>, or zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> 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>^<i>+/+</i> oocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i>^<i>-/-</i> oocytes. (<b>e</b>) γH2AX is largely absent from the synapsed chromosomes of the <i>Mcmdc2</i>^<i>+/+</i> oocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i>^<i>-/-</i> 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>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i> 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>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> oocytes (Mann Whitney test).</p