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Testis |
-H2AX Signaling in the Testis1
a Department of Endocrinology, Faculty of Biology, Utrecht University, 3584 CH Utrecht, The Netherlands
b Departments of Cell Biology
c Radiotherapy, UMCU, 3584 CX Utrecht, The Netherlands
d MCG-Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratory, Leiden University, 2333 AL Leiden, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These -H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, -H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit -H2AX foci but show homogeneous nuclear -H2AX staining, whereas in pachytene spermatocytes -H2AX is only present in the sex vesicle. In response to ionizing radiation, -H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, -H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear -H2AX staining in leptotene spermatocytes demonstrates a function for -H2AX during meiosis. -H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of -H2AX foci at DNA double-strand breaks.
apoptosis, meiosis, signal transduction, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In response to ionizing radiation, DNA double-strand breaks (DSBs) are generated [9]. One of the first steps in the cellular response to DSBs is phosphorylation of histone H2AX at serine 139 [10], and very shortly after irradiation -H2AX (phosphorylated H2AX) foci appear specifically at the damaged sites containing DSBs [11, 12]. At these sites of DNA damage, -H2AX appears to have a critical function in the recruitment of DNA repair factors and DNA damage-signaling proteins [12, 13]. Moreover, H2AX phosphorylation is an early chromosome modification that is followed by apoptotic DNA fragmentation [14] and constitutes an important step in the course of mammalian apoptosis [15]. Recently, a H2AX-/- mouse was generated [16]. Although loss of H2AX did not seem to impair cell cycle checkpoints, it did lead to increased chromosomal abnormalities, deficiencies in gene targeting, impaired DNA repair, and subsequently increased radiosensitivity. Also H2AX-/- cells exhibit increased sensitivity to ionizing radiation and genomic instability [17].
After exposure to ionizing radiation, the tumor suppressor p53 is induced in spermatogonia and plays a central role in DNA damage-induced spermatogonial apoptosis [18–20]. In response to DNA damage, p53 can be activated by members of the large family of phosphatidylinositol-3 kinases including ATM (ataxia telangiectasia mutated) and DNA-PK (DNA-dependent protein kinase), leading to cell cycle arrest or apoptosis [9]. In contrast to some researchers [21, 22], most have found that p53 induction is independent of DNA-PK [23–25], whereas ATM is very important if not essential for p53 activation in response to DNA damage [26–28].
Moreover, histone H2AX is phosphorylated by ATM in response to DSBs, whereas -H2AX foci formation is independent of DNA-PK [15, 29]. After induction of DSBs, a fraction of the nuclear ATM pool relocates to the damaged DNA and colocalizes with -H2AX foci [30]. The p53 binding protein 1 (53BP1) also colocalizes with -H2AX foci and becomes hyperphosphorylated in response to DNA damage in an ATM-dependent manner [13]. These results suggest that -H2AX foci formation could be an early step in spermatogonial p53 activation and thus in spermatogonial apoptosis in response to ionizing radiation.
DSBs are not only induced by ionizing radiation, also V(D)J recombination in lymphoid cells [31, 32], and homologues recombination during meiosis [33] generate DSBs and subsequently -H2AX foci. Loss of H2AX does not affect V(D)J recombination or nonhomologues end-joining (NHEJ) but does affect class-switch recombination [16, 17], indicating that H2AX is involved in other than classical NHEJ DNA repair pathways.
Timing of DSB induction during meiotic recombination has been investigated on surface spreads of spermatogenic cells using immunolocalization of -H2AX and markers specific for certain stages of meiosis [33]. DSBs occurred predominantly during the leptotene stage and thus before synapsis of the homologous chromosomes. During zygotene and throughout the pachytene stage, -H2AX staining became restricted to the sex vesicle that contains the X and Y chromosomes. In H2AX-/- testes, preleptotene and leptotene spermatocytes were normally present, and spermatogenesis was arrested at the pachytene stage of meiosis I. This arrest appeared to be associated with defects in sex chromosome segregation and impaired meiotic crossover [16].
During spermatogenesis both DNA integrity and chromatin organization are essential, and in both processes -H2AX most likely plays a role. We investigated in which germ cells -H2AX foci appear in the testis in response to ionizing radiation, whether spermatogonial p53 induction is correlated with the appearance of -H2AX, and whether DNA-PK is involved in these processes. Furthermore, independent of irradiation, we studied more closely the exact timing of the appearance of -H2AX during spermatogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The testes of male FvB/NAU mice (Central Laboratory Animal Institute, Utrecht, The Netherlands) at least 7 wk of age were locally irradiated with a single dose of 4 Gy of X-rays (200 kV, 20 mA, 0.5-mm Cu filter; Philips, Eindhoven, The Netherlands). Groups of four mice (with three replicates of each experiment) were killed by cervical dislocation at 3, 6, 12, and 24 h after a dose of 4 Gy. Control mice were sham irradiated.
For immunohistochemistry, one testis of each mouse was fixed in 4% phosphate-buffered formaldehyde (pH 6.6–7.2) for 4 h and postfixed in a diluted Bouin solution (71% picric acid [0.9%], 24% formaldehyde [37%], 5% acetic acid) for 16 h at room temperature. Tissues were washed in 70% EtOH prior to embedment in paraffin (Stemcowax; Adamas Instruments, Ameronger, The Netherlands).
Homozygous scid mice (scid/scid) on a CB-17 genetic background [34] and wild-type CB-17 mice were propagated and irradiated as described [34], receiving a dose of 4 Gy of total body X-irradiation. The testes of groups of four mice were fixed in 4% phosphate-buffered formaldehyde (pH 6.6–7.2) or in a diluted Bouin solution at 3 and 12 h after irradiation.
For protein isolation, the contralateral testes were frozen in liquid nitrogen and stored at -80°C. The animals were used and maintained according to regulations provided by the animal ethical committee of the University Medical Center Utrecht, which also approved the experiments.
Immunohistochemistry
Paraffin-embedded sections (5 µm) of testes at different intervals after irradiation were mounted together on a TESPA (3-aminoproyl-tri-ethoxysilane)-coated glass slide and dried overnight at 37°C. Sections were dewaxed in xylene and hydrated in a graded series of alcohols. Between each step, sections were washed in PBS. For p53 staining, the sections were boiled three times for 10 min each in 0.01 M sodium citrate using a microwave oven (H2500; Bio-Rad, Hercules, CA). Sections were then incubated in 0.35% H2O2 in PBS for 10 min. Blocking occurred in 5% BSA (Sigma, St. Louis, MO)/5% goat serum (Aurion, Wageningen, The Netherlands) in PBS. The slides were then incubated with rabbit polyclonal antibodies against -H2AX (antiphospho-H2AX [Ser139]; Upstate Biotechnology, Lake Placid, NY) or rabbit polyclonal anti-p53 antibodies (NCL-p53-CM5p; Novocastra Laboratories Ltd., Newcastle, U.K.) diluted 1:50 in PBS including 1% BSA in a humidified chamber overnight at 4°C. Incubation with secondary biotinylated goat anti-rabbit IgGs (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in PBS including 1% BSA was performed in a humidified chamber for 60 min at room temperature. The horseradish peroxidase avidin-biotin complex reaction was performed according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). Bound antibodies were visualized using 0.3 µg/µl 3,3'-diaminobenzidine (DAB; Sigma) in PBS, to which 0.03% H2O2 was added. Sections were counterstained with Mayer hematoxylin. For negative controls, primary antibodies were replaced by rabbit IgGs. Adjacent sections were used for periodic acid-Schiff staining to identify the stages of the cycle of the seminiferous epithelium. Sections were dehydrated in a series of graded alcohols and xylene and mounted with Pertex (Cellpath Ltd., Hemel Hempstead, U.K.).
Confocal Microscopy
For confocal microscopy, a protocol similar to that for immunohistochemistry was used. For p53 antigen retrieval, the sections were boiled three times for 10 min each time in 0.01 M sodium citrate using an H2500 microwave oven. For double labeling, goat monoclonal antibodies against p53 (M-19, Santa Cruz Biotechnology) were used instead of the rabbit polyclonal antibodies against p53. Fluorescent secondary antibodies were used instead of biotinylated antibodies, and 0.2% gelatine in PBS was used instead of PBS alone. Sections were not incubated in 0.35% H2O2 in PBS but instead were incubated in 50 mM glycine in PBS for 30 min. Fluorescein isothiocyanate-labeled goat anti-rabbit and Texas Red-labeled rabbit anti-goat antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). The sections were mounted in VECTAshield (Vector) and viewed with a 63x planapo objective on a Leitz DMIRB fluorescence microscope (Leica, Voorburg, The Netherlands) interfaced with a Leica TCS4D confocal laser scanning microscope (Leica, Heidelberg, Germany). Images were recorded digitally. Negative control sections were treated in the same way, except that the primary antibodies were omitted.
Western Blot Analysis
Total protein lysates were prepared by homogenizing the testes in a polytron device (Janke & Kunkel GmbH, Staufen, Germany). The cells were then lysed in RIPA buffer (PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM Na3VO4, 1 mM Na2MoD4, and 10 mM NaF) for 30 min on ice. Lysates were sonicated on ice and cleared by centrifugation. Protein levels were measured using bicinchoninic acid analysis (Pierce Chemical Co., Rockford, IL). SDS-PAGE was performed as described by Laemmli [35]. Proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (MilliPore, Bedford, MA).
Western blots were blocked using Blotto-A containing 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) including 0.05% Tween-20 (TBST) and were washed in TBST between each step. First antibodies were diluted 1:1000 in Blotto-A. After incubation with secondary antibodies conjugated to horseradish peroxidase (DAKO A/S, Glostrup, Denmark) diluted 1:5000 in Blotto-A, the antigens were visualized using chemiluminescence (ECL; Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands) and exposure to X-ray film (RX; Fuji Photo Film Co., Tokyo, Japan).
Stripping of blots was performed at 50°C for 30 min in stripping buffer (0.2% SDS, 0.1 M Tris-HCl, 0.01% ß-mercaptoethanol). The stripped blots were reanalyzed using a mouse polyclonal antibody against 
-tubulin (AM-2495-11; InnoGenex, San Ramon, CA).
Coimmunoprecipitations
Volumes of testis lysates equivalent to 0.5 mg of protein were taken from whole testis lysates of sham-irradiated testes and irradiated testes 6 and 24 h postirradiation, and RIPA was added to a total volume of 200 µl with a final percentage of 0.05% SDS. Protein A-agarose beads (50% suspension; Repligen Co., Cambridge, MA) (15 µl/sample) were washed five times with 1% BSA in PBS. Rabbit polyclonal antibodies against -H2AX (10 µl), rabbit polyclonal antibodies against p53 (10 µl), or rabbit IgGs for negative controls (10 µl) were incubated with the beads in 300 µl for 1 h at 4°C. The prepared tissue lysates were precleared with washed beads for 1 h at 4°C. Precleared tissue lysates were then added to the incubated beads or to washed beads for additional negative controls and incubated for 1 h at 4°C in a total volume of 0.5 ml of RIPA. The beads were then washed three times in RIPA and dried using a 30-gauge needle. Finally, 20 µl of Laemli sample buffer was added, and the samples were analyzed (see Western Blot Analysis).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-H2AX in the Testis
Using an antibody against phosphorylated -H2AX, the presence of -H2AX in the testis was studied by immunohistochemistry on sections of sham-irradiated testes and irradiated testes fixed at 1.5–24 h after a dose of 4 Gy of 200-kV X-rays.
In the nonirradiated testis, strong homogeneous nuclear staining and pronounced -H2AX foci were already present in all intermediate and B spermatogonia (Fig. 1A). This staining remained until the zygotene spermatocyte stage. In pachytene spermatocytes, only the sex vesicles were stained (Fig. 1, A and C), and this staining disappeared during the meiotic divisions in stage XII of the cycle of the seminiferous epithelium. The A spermatogonia (Fig. 1B) and round spermatids (Fig. 1, A and D), also exhibited homogeneous nuclear staining, although less strongly. Elongated spermatids (Fig. 1A) and all somatic cells were negative for -H2AX.
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After irradiation, nuclear -H2AX foci also became visible in the A spermatogonia (Fig. 1F), pachytene spermatocytes (Fig. 1G), and round spermatids (Fig. 1H). These differences in -H2AX staining could not be visualized using Western blot analysis; the intensities of the -H2AX bands were the same at all time points after irradiation (Fig. 1I). No -H2AX staining appeared in any of the somatic myoid, Leydig, or Sertoli cells after irradiation. The controls, in which the primary antibodies were replaced with rabbit IgGs, were all negative.
-H2AX Interacts with p53 after Irradiation
The tumor suppressor p53 plays a key role in radiation-induced spermatogonial apoptosis [18–20] and is induced in these cells 3 h after irradiation [18–20]. To investigate whether -H2AX foci formation in response to irradiation is correlated with p53 induction, we coimmunoprecipitated p53 from total testis lysates using antibodies against -H2AX and coimmunoprecipitated -H2AX using antibodies against p53 at several time points after irradiation (Fig. 2). Binding of p53 to -H2AX increased in response to irradiation, with maximum binding between 3 and 6 h after irradiation. Binding of -H2AX to p53 also increased 3 h after irradiation (Fig. 2). When p53 or -H2AX was immunoprecipitated and immunoblotted using antibodies against the same proteins, both p53 and -H2AX were immunoprecipitated and behaved as described previously (Figs. 1I and 2) [18–20]. The negative controls in which the antibodies were omitted or replaced with rabbit IgGs were all negative.
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Interaction Between p53 and -H2AX Is Spermatogonial
The mouse germ cell types in which both -H2AX foci and p53 expression are induced in response to irradiation and that subsequently undergo apoptosis are the spermatogonia. Therefore, the interaction between -H2AX and p53 was expected to be spermatogonial. To study this interaction, we performed a p53/-H2AX double-labeling experiment and confocal microscopy. Both p53 and -H2AX were induced in spermatogonia after irradiation and colocalized in the nucleus (Fig. 3). Although other cell types also express these proteins [18–20] (Fig. 1), colocalization was only found in the irradiated spermatogonia. Even in spermatocytes that also weakly express p53, p53 did not colocalize with the radiation-induced -H2AX foci. These results support the hypothesis that the interaction between p53 and -H2AX found in total testis lysates is spermatogonial.
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-H2AX Foci Formation and p53 Signaling in the Testis Are Independent of DNA-PK
To investigate whether DNA-PK is involved in H2AX phosphorylation and p53 signaling in the testis, we repeated the localization and biochemical experiments using DNA-PKcs-deficient scid mice on a CB-17 genetic background [34].
We compared p53 induction in scid and wild-type testes using sham-irradiated testes and irradiated testes fixed 3 h after a 4-Gy dose of X-rays (after 3 h, p53 is known to be induced in wild-type spermatogonia [18–20]). In the wild-type sham-irradiated testis, weak p53 staining was present in the nuclei of Sertoli cells (Fig. 4A). As expected, 3 h after irradiation nuclear p53 induction was observed in wild-type spermatogonia (Fig. 4C) but also in scid spermatogonia (Fig. 4D). In contrast to the wild-type testes (Fig. 4A), in the scid testes p53 also appeared in some spermatogonia in sham-irradiated testes (Fig. 4B).
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To compare p53 protein levels in the scid testis with those in the wild-type testes, whole testis lysates of sham-irradiated mice and of irradiated mice killed at 3 and 12 h post-irradiation were analyzed using Western blotting. In both the scid and the wild-type testes, p53 levels were elevated 3 h after ionizing radiation, as described previously for wild-type testes. No significant differences were observed between scid and wild-type testes (Fig. 4E). To verify that equal amounts of protein were loaded on the gel, the blots were stripped and reprobed using an antibody against -tubulin (Fig. 4E).
The repeated experiments for -H2AX performed using scid mice all gave exactly the same results as the control experiments using wild-type CB-17 (Figs. 5 and 6) or FvB/NAU (Figs. 1 and 2) mice. In the immunohistochemistry experiments, no differences in -H2AX staining pattern were observed between formalin-fixed tissue and tissue fixed in diluted Bouin solution or between sections that were or were not boiled in 0.01 M sodium citrate for antigen retrieval. These results support the hypothesis that H2AX phosphorylation and the interaction between p53 and -H2AX are independent of DNA-PK.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-H2AX foci in leptotene and early zygotene spermatocytes [33]. However, the present data show that independent of irradiation clear -H2AX foci are already present in all intermediate and B spermatogonia remaining up until zygotene spermatocytes. Additionally, homogeneous nuclear staining is present in type A spermatogonia and round spermatids, whereas in pachytene spermatocytes only the sex vesicles are stained. The expression pattern in the testis contradicts the assumed restriction of -H2AX localization to DSBs during spermatogenesis. Moreover, for somatic cell lines, DSB-independent -H2AX formation occurs in response to replicational stress [36]. After irradiation, -H2AX foci also appeared in type A spermatogonia, pachytene spermatocytes, and round spermatids.
In the testis, -H2AX appeared to interact with the tumor suppressor p53, which plays a key role in spermatogonial apoptosis [18–20]. This interaction increased in response to ionizing radiation. Although recent research has indicated the existence of a -H2AX-p53 pathway [12, 13], the present study is the first in which an interaction between these proteins has been shown. Although spermatocytes weakly express p53 [18, 37], the mouse germ cell types in which both -H2AX and p53 are elevated in response to irradiation (Figs. 1 and 3) and subsequently undergo apoptosis are spermatogonia [18–20]. Therefore, the interaction between -H2AX and p53 was predicted to be confined to spermatogonia. This prediction was confirmed by nuclear colocalization of p53 and -H2AX only in the irradiated spermatogonia. Apparently, spermatogonia and spermatocytes follow different apoptotic pathways, as described in a previous study in which irradiation-induced spermatogonial apoptosis was p53 dependent but the apoptotic elimination of spermatocytes with synaptic errors was p53 independent [38].
As others have described previously for other systems [23–25], we have demonstrated that spermatogonial p53 induction is independent of DNA-PK. We also showed that phosphorylation of H2AX, -H2AX foci formation, and the interaction of p53 with -H2AX in the testis are DNA-PK independent. These results are consistent with those of other studies that have demonstrated DNA-PK independent phosphorylation of H2AX [15, 29] and activation of p53 [23–25]. The most likely candidate for acting upstream of spermatogonial -H2AX foci formation and p53 activation seems to be DNA-PK's close relative ATM. ATM has already been implicated as an important upstream activator of p53 [26–28] and recently was shown to phosphorylate H2AX [29] and to colocalize with -H2AX foci [30].
Based on our and previous results, we propose the following sequence of events in irradiated spermatogonia. ATM phosphorylates H2AX, which forms -H2AX foci at the sites of DSBs. At these sites, -H2AX then recruits DNA repair and damage-response proteins [12], including 53BP1 [13] and p53. ATM then activates p53 [26–28], which induces spermatogonial apoptosis or cell cycle arrest [18–20].
Also during meiotic recombination, DSBs and -H2AX foci are formed [33]. However, in spermatocytes, -H2AX does not colocalize with p53, and in early spermatocytes -H2AX appears independent of irradiation. After irradiation, when -H2AX foci appear in all pachytene spermatocytes and round spermatids (Fig. 1, F and G), these foci do not colocalize with p53 (Fig. 3); irradiation does not lead to apoptosis of these cells [3]. Additionally, irradiation-induced spermatogonial apoptosis is p53 dependent, whereas the apoptotic elimination of spermatocytes with synaptic errors is independent of p53 [38]. Apparently, -H2AX foci can lead to different cellular responses in different types of germ cells.
Using squashed germ cells and molecular markers, Mahadevaiah et al. [33] demonstrated that -H2AX foci appear in preleptotene and leptotene and disappear in zygotene spermatocytes; they concluded that meiotic DSBs precede recombinational synapsis. However, using immunohistochemistry, we demonstrated that in the nonirradiated testis -H2AX foci are already present in intermediate and B spermatogonia (Fig. 1A). If -H2AX is a marker for DSBs, then DSBs must remain unrepaired during two mitotic divisions or must be formed anew after each division, until the B spermatogonia divide into preleptotene spermatocytes. In this context, even -H2AX staining in preleptotene spermatocytes [33] is controversial, because these cells represent the premeiotic S phase and are replicating their DNA to become 4C leptotene spermatocytes [39]. Although we found -H2AX already present earlier in spermatogenesis, our results do not contradict the conclusion of Mahadevaiah et al. [33] that DSBs precede recombinational synapsis, which is supported by experiments with Spo11-/- mice. Meiotic DSBs are assumed to be generated by Spo11 [40, 41], but although it disappears from leptotene and zygotene spermatocytes, -H2AX staining remains in Spo11-/- preleptotene spermatocytes [33]. These results are consistent with our finding of H2AX phosphorylation before the leptotene stage when recombinational DSBs are induced. The detection or lack of detection [33] of -H2AX in intermediate and B spermatogonia is most likely due to differences between the histological and cytological approaches. The strong -H2AX staining of the sex vesicle throughout the pachytene stage [33] (Fig. 1, C and G) would be inexplicable if -H2AX were only present at DSBs. In the H2AX-/- testis, the sex chromosomes fail to pair and are fragmented or associated with autosomal chromosomes [16], indicating a specific function for H2AX in the sex vesicle.
Dividing cells in culture form -H2AX foci at DSBs [10–12]. However, in the testis various quiescent somatic cell types, such as Sertoli cells, Leydig cells, myoid cells, and blood cells, do not form -H2AX foci, even after treatment with ionizing radiation, although DSBs are also introduced in these cells. Moreover, these cells do not induce p53 in response to irradiation [18–20] nor do they undergo apoptosis. Hence, in the testis different cell types respond differently to DSBs, and the absence of -H2AX foci and p53 in the quiescent testicular cells very likely represents a general feature of quiescent cells in vivo.
A histone H2A variant from the mouse testis has been characterized as similar to H2AX [42], and the appearance of -H2AX very much coincides with the expression of this nonphosphorylated H2A in the testis [43]. The absence of -H2AX in Leydig cells could thus be explained, because these cells express H2A only very weakly. However, the nonphosphorylated H2A is distinctively expressed in Sertoli cells, again indicating that even the different somatic cell types in the testis display different chromatin organization and responses to DNA damage.
These findings raise the question of whether H2AX phosphorylation in the testis is a marker for DSBs. During spermatogenesis, chromatin is constantly being reconstructed [44]. These changes, such as altering DNA methylation, start in the mitotic spermatogonia [45]. The major morphological difference between A spermatogonia and intermediate and B spermatogonia is the appearance of heterochromatin in the nucleus [6–8]. During the remodeling of euchromatin to heterochromatin, methylation and deacetylation of histones play a key role [46], and in intermediate and B spermatogonia H2AX phosphorylation may also be involved. In pachytene spermatocytes, when most of the linker H1 histones are replaced by the testis-specific subtype H1t [44], -H2AX appears to have an essential function in the sex vesicle [16, 33] (Fig. 1). Histone ubiquitination is needed for chromatin modifications that occur at this stage and during the histone-to-protamine replacement in haploid spermatids [47, 48], whereas a function for histone phosphorylation at these stages has never been considered. The currently known facts about histones and spermatogenesis in combination with our results indicate that the function of H2AX phosphorylation in the testis is not restricted to DSBs.
In response to ionizing radiation, -H2AX foci appear, and in spermatogonia the interaction between -H2AX and p53, which induces spermatogonial apoptosis, increases. Independent of irradiation, -H2AX is thought to mark sites of meiotic DSBs [33]. However, during spermatogenesis, -H2AX also has other functions that do not necessarily involve DSBs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Geert Hamer, Department of Endocrinology, Faculty of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; g.hamer{at}bio.uu.nl
Received: 25 June 2002.
First decision: 18 July 2002.
Accepted: 28 August 2002.
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REFERENCES |
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Y. Tadokoro, K. Yomogida, Y. Yagura, S. Yamada, M. Okabe, and Y. Nishimune Characterization of Histone H2A.X Expression in Testis and Specific Labeling of Germ Cells at the Commitment Stage of Meiosis with Histone H2A.X Promoter-Enhanced Green Fluorescent Protein Transgene Biol Reprod, October 1, 2003; 69(4): 1325 - 1329. [Abstract] [Full Text] [PDF]
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 S.-R. Yoon, L. Dubeau, M. de Young, N. S. Wexler, and N. Arnheim Huntington disease expansion mutations in humans can occur before meiosis is completed PNAS, July 22, 2003; 100(15): 8834 - 8838. [Abstract] [Full Text] [PDF]
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G. Hamer, H. L. Roepers-Gajadien, A. van Duyn-Goedhart, I. S. Gademan, H. B. Kal, P. P.W. van Buul, T. Ashley, and D. G. de Rooij Function of DNA-Protein Kinase Catalytic Subunit During the Early Meiotic Prophase Without Ku70 and Ku86 Biol Reprod, March 1, 2003; 68(3): 717 - 721. [Abstract] [Full Text] [PDF]
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