p53 and TAp63 participate in the recombination-dependent pachytene arrest in mouse spermatocytes

<div><p>To protect germ cells from genomic instability, surveillance mechanisms ensure meiosis occurs properly. In mammals, spermatocytes that display recombination defects experience a so-called recombination-dependent arrest at the pachytene stage, which relies on the MRE11 complex—ATM—CHK2 pathway responding to unrepaired DNA double-strand breaks (DSBs). Here, we asked if p53 family members—targets of ATM and CHK2—participate in this arrest. We bred double-mutant mice combining a mutation of a member of the p53 family (p53, TAp63, or p73) with a <i>Trip13</i> mutation. <i>Trip13</i> deficiency triggers a recombination-dependent response that arrests spermatocytes in pachynema before they have incorporated the testis-specific histone variant H1t into their chromatin. We find that deficiency for either p53 or TAp63, but not p73, allowed spermatocytes to progress further into meiotic prophase despite the presence of numerous unrepaired DSBs. Even so, the double mutant spermatocytes apoptosed at late pachynema because of sex body deficiency; thus p53 and TAp63 are dispensable for arrest caused by sex body defects. These data affirm that recombination-dependent and sex body-deficient arrests occur via genetically separable mechanisms.</p></div

Signaling pathway leading to the activation of the recombination-dependent arrest.

<p>Based on the information presented in this and previous studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006845#pgen.1006845.ref036" target="_blank">36</a>], we propose the following model of the signaling pathway required to activate the recombination-dependent arrest in mouse spermatocytes. DSBs are sensed by the MRE11 complex and lead to activatation of ATM, which in turn activates CHK2, leading to the upregulation of p53 and TAp63, which implement the recombination-dependent arrest that blocks progression to mid/late pachynema.</p

Absence of p53 or TAp63 allows TRIP13-deficient spermatocytes to accumulate H1t despite having multiple unrepaired DSBs.

<p>(A) Schematic representation of arrest events and H1t incorporation from early pachynema to diplonema. (B) Graph showing the average percentage of spermatocytes that have incorporated H1t for each genotype and error bars represent standard deviation (SD). (C-J) Spread chromosomes from representative spermatocytes of the indicated genotypes and stages, stained for the axial element protein SYCP3 (green), H1t (blue), and γH2AX (red). The large, bright blobs of γH2AX staining are sex bodies (arrows); the smaller γH2AX patches reflect unrepaired DSBs (orange arrowheads). Scale bar in J represents 10 μm and applies to all panels. (K) Quantification of the number of γH2AX patches per spermatocyte. Horizontal lines represent means. Means (± SD) are indicated above the graph. Above the means, (n.s.) indicates not significant and (*) indicates significantly different relative to <i>Trip13</i>^{<i>mod/mod</i>}. (Statistical tests and p values are stated in main text). The total number of cells studied (n) and the number of analyzed animals per genotype (N) are shown in each graph.</p

Apoptosis tends to occur at a later (H1t-positive) stage in TRIP13-deficient spermatocytes lacking p53 or TAp63.

<p>(A-H) Representative apoptotic (TUNEL-positive) pachytene-stage spermatocytes stained for SYCP3 and TUNEL (both in green), H1t (blue), and γH2AX (red). Note the presence of multiple γH2AX patches and abnormal sex body in the apoptotic, H1t-positive spermatocyte from the <i>Trip13</i>^{<i>mod/mod</i>} <i>p53</i>^<i>-/-</i> mutant. Scale bar in H represents 10 μm and applies to all panels. (I) Percentage of apoptotic spermatocytes that are H1t-negative or H1t-positive. Error bars represent SD. The total number of cells studied (n) and the number of analyzed animals per genotype (N) are shown. Note that error bars from <i>Trip13</i>^{<i>mod/mod</i>} <i>p53</i>^<i>-/-</i> mutant are almost too small to be seen.</p

Mutation of <i>p53</i> does not rescue <i>Spo11</i>^<i>-/-</i> meiotic arrest.

<p>(A,B) Representative tubule sections from adult testes stained with PAS-Hematoxylin. Scale bar in B represents 20 μm and applies to panels A and B. (C,D) Spermatocytes stained for SYCP3 (green), γH2AX (red), and DAPI (blue). Note the presence of a pseudo-sex body in both cells (arrowheads). Scale bar in D represents 10 μm and applies to panels C and D. (E) Graph displaying the mean percentage of <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i><i>p53</i>^<i>-/-</i> spermatocytes containing a pseudo-sex body. Error bars represent SD. N shows the number of mice analyzed.</p

p53 is up-regulated in TRIP13-deficient pachytene spermatocytes.

<p>Representative testis sections from wild type (A), <i>Trip13</i>^{<i>mod/mod</i>} (B), and <i>Trip13</i>^{<i>mod/mod</i>} <i>TAp63</i>^<i>-/-</i> (C) stained with DAPI and for p53 are shown. Cross sections of tubules of different epithelial stages are shown to illustrate staining patterns during progression of spermatocytes from leptonema to pachynema. Insets show marked spermatocytes from different stages. Note that the presence of all spermatogenic cells allows for the precise staging of the tubules in wild type samples. However, the absence of most secondary spermatocytes and spermatids impedes an accurate staging of <i>Trip13</i> mutant tubule sections. Nonetheless, tubules in which spermatocytes make up the outermost layer of cells can be clearly assigned to stages IX-XII or I. Such tubules contain in the most exterior layer of cells spermatocytes from leptonema to early pachynema. Labels on the panels stand for: Se: Sertoli cell, LSp: leptotene spermatocyte, L/ZSp: leptotene/zygotene spermatocyte, P/DSp: pachytene/diplotene spermatocyte, Sp: spermatocyte, Sd: spermatid. Scale bar represents 20 μm and applies to all panels.</p

Absence of p53 family members does not alleviate spermatocyte apoptosis at epithelial stage IV in TRIP13-deficient mice.

<p>(A–C) Representative tubules are shown from testis sections of the indicated genotypes stained by TUNEL to detect apoptotic spermatocytes (green). Scale bar in A represents 40 μm and applies to panels A–C. (D) Quantification of the percentage of tubules with 0, 1–10, or more than 10 TUNEL-positive cells. The total number of tubules studied (n) and the number of analyzed animals per genotype (N) are shown. (E–I) Representative tubule sections from adult testes stained with PAS-Hematoxylin. Scale bar in I represents 20 μm and applies to panels E–I. (E) Wild-type seminiferous tubule at stage IV containing spermatogonia, spermatocytes and spermatids. (F–I) <i>Trip13</i>^{<i>mod/mod</i>} single mutant and p53 family member double mutant tubule sections showing condensed spermatocytes (apoptotic, orange arrowheads). These tubules can be assigned to stage IV on the basis of the presence of both intermediate (In) and B-type spermatogonia (Sg) along with a single layer of spermatocytes, which are a mix of apoptotic and non-apoptotic pachytene cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006845#pgen.1006845.ref050" target="_blank">50</a>].</p

Premature p63 up-regulation in response to TRIP13 deficiency.

<p>Representative testis sections from wild type (A), <i>Trip13</i>^{<i>mod/mod</i>} (B), and <i>Trip13</i>^{<i>mod/mod</i>} <i>p53</i>^<i>-/-</i> (C) stained with DAPI and for p63 are shown, organized as for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006845#pgen.1006845.g003" target="_blank">Fig 3</a>. Note that in <i>Trip13</i> mutants (B-C), p63 signal appears in the outermost layer of cells of tubules that are at stage IX-XII or I, which corresponds to leptotene to early pachytene stage spermatocytes (sp). In wild-type tubules, early pachytene cells in stage I tubules show no staining for p63, which only appears in pachtyene spermatocytes (Psp) in stage II-III tubules onwards. Thus, p63 expression is prematurely upregulated in <i>Trip13</i> mutant testis. Labels on the panels stand for: Se: Sertoli cell, LSp: leptotene spermatocyte, L/ZSp: leptotene/zygotene spermatocyte, P/DSp: pachytene/diplotene spermatocyte, Sp: spermatocyte, Sd: spermatid. Scale bar represents 20 μm and applies to all panels. Scale bar represents 20 μm and applies to all panels.</p

Sex body defects and MSCI failure in TRIP13-deficient cells lacking p53 or TAp63.

<p>(A–C) Representative H1t-positive pachytene spermatocytes of the indicated genotypes stained for SYCP3 (green), γH2AX (red), and H1t (blue). (D–I) Representative pachytene spermatocytes of the indicated genotypes stained for SYCP3 (green), DAPI (blue), and either ATR (D–F) or SUMO-1 (G–I) (red). Scale bar in C represents 10 μm and applies for panels A–I. White arrows indicate sex chromosomes. (J,K) RNA-FISH performed on <i>Trip13</i>^{<i>mod/mod</i>} <i>p53</i>^<i>-/-</i> spermatocytes. Images display <i>Scml2</i> or <i>Zfx</i> RNA-FISH signal (white arrows), DAPI (blue), and immunostaining for TOPBP1 (green) and γH2AX (red). Insets show zoomed images of the sex bodies. Scale bar on K represents 10 μm and applies to panels J-K. (L,M) Graphs showing percentage of early pachytene spermatocytes expressing <i>Scml2</i> (L) or <i>Zfx</i> (M) RNA-FISH signal. Wild type and <i>Trip13</i>^{<i>mod/mod</i>} data are from ref. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006845#pgen.1006845.ref036" target="_blank">36</a>]. Error bars represent SD.</p

Additional file 2: Table S2. of N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration

Primer sequences used for qRT-PCR, RACE, siRNAs, and vector construction in this study. (XLSX 11 kb