| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis |
a Department of Endocrinology, Faculty of Biology, Utrecht University, 3584 CH Utrecht, The Netherlands
b Department of Cell Biology, UMCU, 3584 CX Utrecht, The Netherlands
c MCG-Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratory, Leiden University, 2333 AL Leiden, The Netherlands
d Department of Radiotherapy, UMCU, 3584 CX Utrecht, The Netherlands
e Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
apoptosis, meiosis, signal transduction, spermatogenesis, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Apart from its function during rearrangement of the genes coding for antigen receptor molecules in lymphoid cells, DNA-PK is also involved in the cellular response to DNA damage, such as caused by ionizing radiation. The Ku heterodimer binds to single- and especially double-stranded DNA breaks (DSBs) followed by binding and activation of DNA-PKcs, which then plays a key role in nonhomologous end joining of the damaged DNA [2–6].
In the adult mouse testis, the mitotically active spermatogonia are the cells most sensitive to ionizing radiation, whereas spermatocytes, which undergo meiotic cell divisions, and spermatids, which develop into spermatozoa, are more resistant [7–11].
The tumor suppressor p53 is induced in spermatogonia after irradiation, and this protein plays a central role in DNA damage-induced spermatogonial apoptosis [12–14]. Despite the fact that DNA-PKcs also has been shown to be an upstream activator of p53 [15, 16], we found p53 induction to occur independent of DNA-PKcs [17], which is in line with several studies reporting DNA-PKcs-deficient cells to have a normal response to ionizing radiation with respect to p53 activation and cell cycle arrest [18–20].
However, during the early prophase of meiosis, from leptotene until early pachytene, breaks associated with meiotic synapsis and recombination [21] often also result in activation of proteins, such as ATM, that are normally activated in mitotic cells after DNA damage [22]. This situation could also be true for DNA-PKcs, although Ku70 has been described not to be expressed in leptotene and zygotene spermatocytes [23]. The absence of Ku70 at this stage might be very important in preventing nonhomologous end joining instead of meiotic homologous recombination. To understand more about the processing of DSBs during meiotic recombination, it is important to know exactly when and where DNA-PKcs and both the Ku proteins are expressed during spermatogenesis.
We studied expression and localization of Ku70, Ku86, and DNA-PKcs in the mouse testis before and after irradiation. Furthermore, using DNA-PKcs-deficient scid mice [24, 25], we examined spermatogonial apoptosis after irradiation and spontaneous apoptosis of spermatocytes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The testes of groups of four male FvB/NAU mice at least 7 wk of age were irradiated and fixed at various time intervals after treatment with a dose of 4 Gy of 200 kV X-rays as described previously [14, 26].
Homozygous scid mice (scid/scid) on a CB-17 genetic background [27] and wild-type CB-17 mice were propagated and irradiated as described [27], receiving a dose of 4 Gy of total body X-irradiation. Groups of four mice were killed by cervical dislocation at 3 and 12 h after irradiation, and one testis of each mouse was fixed using a diluted Bouin fixative [14, 26] or overnight fixation in 4% phosphate-buffered (pH 6.6–7.2) formaldehyde, respectively. Control mice were sham-irradiated.
As described [14, 26], contralateral testes were used for protein isolation. The animals were used and maintained according to regulations provided by the animal ethical committee of the University Medical Center of Utrecht, which also approved the experiments.
Immunochemistry
Immunohistochemistry and Western blot analysis were performed as described previously [14, 26] using goat polyclonal Ku70 (M-19; Santa Cruz Biotechnology, Santa Cruz, CA), goat polyclonal Ku86 (M-20; Santa Cruz Biotechnology), rabbit polyclonal DNA-PKcs (H-163; Santa Cruz Biotechnology), and goat polyclonal DNA-PKcs (C-19; Santa Cruz Biotechnology) antibodies. For negative control sections, one volume of primary antibodies was incubated with five volumes of blocking peptide (Santa Cruz Biotechnology) for 2 h. Adjacent sections were used for periodic acid-Schiff (PAS) staining to identify the stages of the cycle of the seminiferous epithelium.
TUNEL Analysis
TUNEL analysis was performed on 5-µm paraffin-embedded sections of formalin-fixed scid or CB-17 testes according to the manufacturer's protocol (In Situ Cell Death Detection Kit, POD; Roche Diagnostics GmbH, Mannheim, Germany) [14]. Adjacent sections were used for PAS staining to identify the stages of the cycle of the seminiferous epithelium.
For every stage of the seminiferous epithelium, the number of apoptotic spermatogonia/spermatocytes of at least 10 tubule cross sections were counted for each mouse. The average numbers of apoptotic cells for four mice are depicted in Figure 2. Differences between wild-type and scid mice were analyzed using the nonparametric Mann-Whitney test (two-tailed P < 0.05).
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The localization of DNA-PKcs was studied using immunohistochemistry on sections of sham-irradiated testes and testes fixed at 1.5–24 h after a dose of 4 Gy of 200 kV X-rays. Intense, nuclear staining was found in spermatogonia, preleptotene spermatocytes, and pachytene spermatocytes (Fig. 1a). All germ cells and somatic cells, including Leydig and Sertoli cells, showed a weak cytoplasmic staining for DNA-PKcs (Fig. 1, a and b). This staining however also appeared in the negative controls (Fig. 1b), as did Golgi-like staining in round and elongated spermatids. The expression pattern did not change in response to irradiation. Negative controls were performed using antibodies that were preincubated with blocking peptide.
|
Expression of Ku70 and Ku86 in the Testis
Ku70 and Ku86 exhibited similar expression patterns (Fig. 1, c and d). All cells showed a light cytoplasmic staining that also appeared in the negative controls. A stronger cytoplasmic staining, however, was seen in Leydig cells, whereas Sertoli cells showed nuclear staining. A-spermatogonia also showed nuclear staining. However, intermediate spermatogonia and B-spermatogonia appeared negative for the Ku proteins. Staining reappeared in pachytene spermatocytes of stage IV of the cycle of the seminiferous epithelium. Round but not elongating spermatids showed nuclear staining. The expression patterns of Ku70 and Ku86 did not change in response to irradiation.
Thus, all components of the DNA-PK complex are present in the nuclei of type A spermatogonia and pachytene spermatocytes from stage V onward, whereas early spermatocytes express only DNA-PKcs and round spermatids express only the Ku proteins.
Protein Levels of DNA-PK Do Not Change in Response to Irradiation
Because immunohistochemistry is not suitable for quantification of antigens in sections, possible changes in protein levels in the testis before and after irradiation were studied using Western blot analysis of whole testis lysates of sham-irradiated mice and of mice killed 1.5–24 h after irradiation. Ku70, Ku86, and DNA-PKcs levels did not change after X-irradiation (Fig. 1e).
Apoptosis in the Scid Testis
We studied the role of DNA-PK in apoptosis by comparing the number of apoptotic spermatogonia and spermatocytes before and 12 h after X-irradiation in scid and wild-type testes. Normally, after 12 h the most apoptotic spermatogonia are present [14], whereas after 24 h most spermatogonia have disappeared [9, 10]. In the irradiated wild-type testis, many spermatogonia were TUNEL stained (Fig. 2, c and e). However, 12 h after irradiation, in scid testes the apoptotic response to irradiation of spermatogonia appeared much more severe (Fig. 2, d and e). Even Sertoli cells, which phagocytize apoptotic bodies, were often TUNEL stained in the scid testis (Fig. 2d), a response that was especially evident in stages V–VII of the cycle of the seminiferous epithelium, where most apoptotic spermatogonia were observed (Fig. 2e). There were some apoptotic spermatogonia in the nonirradiated scid testis, but compared with the irradiated testes their numbers were negligible. Hence, 12 h after irradiation, the increase in apoptotic spermatogonia is higher in the scid testis.
In sham-irradiated testes of both wild-type [14] and scid mice, very few TUNEL-positive spermatogonia were found (Fig. 2, a and b). In scid testes, however, much higher numbers of apoptotic spermatocytes in early meiotic prophase were found than in the wild type (Fig. 2, b and f). The great majority of these apoptotic spermatocytes were present in stage IV of the cycle of the seminiferous epithelium (Fig. 2f).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
In spermatogonia, DNA-PK detects and assists in repair of DSBs caused by ionizing radiation. In the scid testis, these DNA breaks remain unrepaired, and the persistent DSBs probably activate ATM, another DSB-associated protein that can induce and activate p53 [28–30] and subsequently spermatogonial apoptosis [12–14]. In wild-type spermatogonia, many DSBs are more efficiently repaired, resulting in a lower number of apoptotic cells per tubule cross section 12 h after irradiation (Fig. 2e).
Early spermatocytes, until stage IV pachytene, also express DNA-PKcs but do not express the Ku proteins (Figs. 1 and 3). Until now, Ku-independent DNA binding and activation of DNA-PKcs has been described in vitro only [31]. However, the significantly higher number of spontaneously apoptotic early spermatocytes in the scid testes (Fig. 2f) suggests that in the normal testis DNA-PKcs has a function in early spermatocytes that does not include the Ku heterodimer and is not influenced by ionizing radiation. DNA-PK suppresses homologous recombination [32], which would seriously interfere with meiotic recombination. Although this suppression depends on functional DNA-PKcs [32], DNA-PKcs remains present (Fig. 1a) and functional (Fig. 2f) in early spermatocytes. These results suggest that the complete DNA-PK holoenzyme is required for suppression of homologous recombination. To prevent suppression of meiotic recombination, the Ku proteins are very specifically downregulated in early spermatocytes (Figs. 1, c and d, and 3). This hypothesis is supported by findings that interruption of the DNA-PK pathway stimulates homologous recombination [33, 34].
Furthermore, round spermatids express Ku70 and Ku86 but lack DNA-PKcs (Figs. 1 and 3). Ku86 plays a key role in function and maintenance of the telomeres [35, 36], and in round spermatids the Ku proteins, without DNA-PKcs, might keep telomeric fusions from occurring in the germ line.
Although nonhomologous end joining plays a dominant role in DNA repair during the G1 to early S phase, its role during the late S to G2 phase is only minor [37]. Since nonhomologous end joining would also interfere with proper meiotic recombination, it is interesting that during the meiotic prophase chromosomal radiosensitivity seems comparable with that during the somatic G2 phase [27].
Whereas DNA-PK activity during the G1 to early S phase has been described as correlated with ionizing radiation and DSB repair, its activity during the G2 phase is more likely required for exit from a DNA damage-induced G2 checkpoint arrest [38]. Without DNA-PKcs, many spermatocytes undergo apoptosis at stage IV of the seminiferous epithelium cycle. This finding confirms and extends the notion that a meiotic checkpoint exists at stage IV, when spermatocytes with damaged DNA are induced to undergo apoptosis. At this epithelial stage, spermatogenesis abrogates in several knockout mice, including ATM [39] (unpublished data) and Msh5 [40]. DSBs are associated with meiotic synapsis and recombination [21] and are not efficiently repaired in scid spermatocytes, resulting in more apoptosis and failure to pass epithelial stage IV.
Hence, in the testis, DNA-PKcs has a dual function: in the mitotic and radiosensitive spermatogonia it cooperates with the Ku heterodimer in order to repair radiation-induced DSBs, whereas during the meiotic prophase it appears to guard genome integrity as early spermatocytes undergo meiotic synapsis and recombination and is not influenced by ionizing radiation and independent of the Ku heterodimer.
|
FOOTNOTES |
|---|
2 Correspondence: Geert Hamer, Department of Endocrinology, Faculty of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; e-mail: g.hamer{at}bio.uu.nl
Received: 28 June 2002.
First decision: 23 July 2002.
Accepted: 22 August 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
H2AX in the testis. Biol Reprod 2003 68:628-634 First published on October 23, 2002; 10.1095/biolreprod.102.008672This article has been cited by other articles:
|
|
 |
|
  R. S. Maser, K.-K. Wong, E. Sahin, H. Xia, M. Naylor, H. M. Hedberg, S. E. Artandi, and R. A. DePinho DNA-Dependent Protein Kinase Catalytic Subunit Is Not Required for Dysfunctional Telomere Fusion and Checkpoint Response in the Telomerase-Deficient Mouse Mol. Cell. Biol., March 15, 2007; 27(6): 2253 - 2265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-G. Wang, C. Nnakwe, W. S. Lane, M. Modesti, and K. M. Frank Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase J. Biol. Chem., September 3, 2004; 279(36): 37282 - 37290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Pinon-Lataillade, C. Masson, J. Bernardino-Sgherri, V. Henriot, P. Mauffrey, Y. Frobert, S. Araneda, and J. F. Angulo KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis J. Cell Sci., July 15, 2004; 117(16): 3691 - 3702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Hamer, H. B. Kal, C. H. Westphal, T. Ashley, and D. G. de Rooij Ataxia Telangiectasia Mutated Expression and Activation in the Testis Biol Reprod, April 1, 2004; 70(4): 1206 - 1212. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
