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BOR - Papers in Press, published online ahead of print November 26, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022541
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BIOLOGY OF REPRODUCTION 70, 910–918 (2004)
DOI: 10.1095/biolreprod.103.022541
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Transient DNA Strand Breaks During Mouse and Human Spermiogenesis:New Insights in Stage Specificity and Link to Chromatin Remodeling1

Ludovic Marcon, and Guylain Boissonneault2

Department of Biochemistry, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.

gamete biology, gametogenesis, spermatid, spermatogenesis, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian spermiogenesis involves important changes in cytoarchitecture and a dramatic remodeling of the somatic chromatin with the elimination of most of the nucleosomal DNA supercoiling [1, 2]. The modification in chromatin structure occurring in elongating spermatids emerges as an important contribution to the nuclear integrity and acquisition of the full fertilizing potential of the male gamete. In mouse models, deficiency in the expression of the major spermatidal nuclear proteins such as the transition proteins (TPs) or protamines resulted in alteration of sperm chromatin condensation with reduction of fertility [3–5]. Not surprisingly, the predictive value of sperm chromatin integrity for pregnancy outcome has been demonstrated and emerges as a new sperm parameter in the clinical setting [6, 7].

In addition to the nuclear protein exchange, the chromatin remodeling process involves the elimination of the free DNA supercoils created by the nucleosome withdrawal [2, 8]. This dramatic modification in DNA topology must require DNA strand breaks to provide the necessary swivel effect to relieve torsional stress. Consistent with this hypothesis, DNA strand breaks have been previously observed at midspermiogenesis steps in both rat [9, 10] and mouse using in situ nick translation [11] or the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) [12]. In these cases, however, it was not clear whether this was a general process, as only a fraction of the whole population of elongating spermatids was shown to be labeled. To sustain a global change in DNA topology, one would expect that close to 100% of the spermatids must undergo strand breakage. In one unique report, evidence was presented that the vast majority of elongated spermatids near the seminiferous tubule lumen where labeled by the TUNEL assay [13]. Labeling occurred in 20–40% of the tubules, suggesting the stage-specific appearance of the strand breaks. Clearly, a more careful assessment of the extent and stage-specificity of the transient DNA strand breaks in elongating spermatids is required as an essential first step to further characterize what may prove to be a sensitive process for the DNA integrity of the male gamete.

Given the similarity in spermatidal chromatin remodeling among mammals [14, 15], it is likely that the human spermatids undergo such a transient DNA strand breakage and repair process. In light of the reported difficulties in assisted reproductive technologies making use of spermatids for intracytoplasmic sperm injections (ICSI) and the potential genetic risk associated with DNA fragmentation [16–18], it was therefore crucial to investigate whether such a transient appearance of DNA strand breaks may be found in the human spermatids.

Using a sensitive fluorescent TUNEL assay on testis sections, we demonstrate that not only a fraction but the whole population of elongating spermatids in mice harbors DNA strand breaks at steps coincident with the chromatin remodeling or stages IX–XI of the seminiferous epithelium cycle. We also demonstrate for the first time that DNA strand breaks are also detected during the spermatidal chromatin remodeling process in human.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental procedures were approved by the Université de Sherbrooke Ethical Committee for animal experimentation and were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

Animals and Tissue Preparation

Adult male CD-1 mice were obtained from Charles River Breeding Laboratory (St-Constant, PQ, Canada) and killed by CO2 asphyxiation. Freshly excised testes and epididymides were fixed directly in Carnoy (ethanol, chloroform, acetic acid 6:3:1) at 4°C overnight and then embedded in paraffin according to standard procedures. Five µm-thick sections were mounted on Superfrost plus glass slides (Fisher Scientific, St-Laurent, QC).

Human Testicular Biopsies

Sections of testicular biopsies from fertile men were kindly provided by Dr. R. Sullivan (Université Laval, Sainte-Foy, PQ, Canada). Testis biopsies were immediately fixed in 4% buffered paraformaldehyde at 4°C overnight, and samples were then dehydrated, cleared in xylene, and embedded in paraffin. Five µm-thick sections were obtained using a Leica RM 2135 rotative microtome (Leica Microsystems, Wetzlar, Germany). All biopsies were obtained after informal consent from the patient and approval from the Medical Ethics Committee of the Laval University Hospital Center.

Antibodies

The mouse monoclonal HupN1 antiserum raised against purified protamine 1 was a generous gift from Dr. R. Balhorn (Lawrence Livermore National Laboratory, Livermore, CA). Rabbit polyclonal antibody against hyperacetylated histone H4 was purchased from Upstate Biotechnology (Lake Placid, NY).

Immunofluorescence Microscopy on Testis Sections

Paraffin-embedded sections were deparaffinized with xylene, rehydrated through a graded serie of ethanol solutions, washed with water, and equilibrated 5 min in phosphate-buffered saline (PBS). After deparaffinization, sections were blocked in PBS, pH 7.4, containing 1% (w/v) bovine serum albumin and 0.1% Triton X-100 for 30 min at room temperature. The slides were then incubated overnight at 4°C in a humidified chamber with the primary antibody. The sections were then washed three times for 5 min in PBS and incubated with Rhodamine-conjugated secondary antibodies (either goat anti-mouse or goat anti-rabbit IgG according to the species in which the primary antibody has been raised; Bio/Can Scientific, Mississauga, ON, Canada). Following three additional washes, sections were then counterstained and mounted with coverslips using Vectashield mounting medium containing 4,'6'-diaminido-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Indirect immunofluorescence was examined using a Zeiss Axioscop 2 microscope equipped with 10x and 40x objectives (Carl Zeiss Inc., Oberkochen, Germany) and a Spot cooled color digital camera (Diagnostic Instruments Inc., St. Sterling Heights, MI). For negative controls, sections were processed as described above except that the primary antibody was omitted. Stages of the mouse seminiferous epithelium were determined based on previously established morphological criteria [19] whereas human testis sections were staged based on criteria originally described by Clermont [20].

Terminal Deoxynucleotidyl Transferase dUTP-BiotinNick-End Labeling Assay

DNA strand breaks on mouse and human testis sections were detected using a modified TUNEL assay based on protocol previously described in Sun et al. [21]. Briefly, tissue sections were deparaffinized and rehydrated as described above. After three washes in PBS, sections were preincubated inside a humidified chamber with TdT buffer (Roche Diagnostics, Laval, PQ, Canada) for 30 min at 37°C. The TdT buffer was discarded, replaced by fresh buffer (50 µl) containing 25 U terminal transferase and 0.5 nmol Biotin-16-dUTP (Roche Diagnostics) and allowed to incubate for 60 min at 37°C. The end-labeling reaction was stopped by washing the sections with PBS. The sections were then incubated with fluorescein-avidin DN (Vector Laboratories) diluted 1:100 in PBS for 60 min at room temperature to reveal biotinylated UTP extension of free 3'-OH ends. After incubation, the slides were washed three times in PBS and finally mounted with coverslips using Vectashield mounting medium containing DAPI. Counts of TUNEL-positive spermatids were made on at least five seminiferous tubules cross sections at each stage and normalized to total spermatids counts as determined by DAPI staining. Negative control sections were processed without the TdT enzyme added to the TUNEL mixture. TUNEL assay on mice epididymides and human testis sections were carried out using the same protocol and slides were mounted with Vectashield containing DAPI to counterstain DNA.

Double Labeling

For the double detection of DNA strand breaks and nuclear proteins (AcH4, P1), testis sections were first treated for immunofluorescence and then processed for TUNEL assay as described above. Following incubation with the TUNEL mixture and fluorescein-avidin solution, the slides were washed in PBS (3x 5 min) and mounted with Vectashield containing DAPI.

Sperm Decondensation Control

Mice epididymides were dissected out, minced in a Petri dish in ice-cold PBS, pH 7.4, using a sterile razor blade. The resulting cell suspension was filtered through an 80-µm nylon mesh and centrifuged at 2500 rpm, 5 min at 4°C. The pellet was resuspended and centrifuged again. The pellet, containing mainly epididymal sperm, was resuspended in cold PBS containing 2 mM dithiothreitol and 0.5% Triton X-100 and incubated 30 min at room temperature [22]. An equal volume of 4 M NaCl solution was then added to the epidydimal sperm suspension. Fifty µl of the suspension were applied onto poly-L-lysine-coated glass slides, incubated 10 min at room temperature, washed 3 times in PBS 5 min, and then submitted to the TUNEL assay as described above. The slides were mounted in Vectashield mounting medium containing DAPI and examined by fluorescence microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stage-Specific DNA Strand Breaks in Mouse Spermatids

To assess the presence of germ cell-specific DNA strand breaks during mammalian spermiogenesis, we performed a sensitive TUNEL assay on paraffin-embedded cross sections of adult mouse testis. As shown in Figure 1, A and B, endogenous DNA strand breaks were found to be restricted to elongating spermatids and early spermatocytes seen as a row of TUNEL-positive cells at the basal compartment of the seminiferous tubules. Examination of a large number of tubules confirmed that labeling of the early spermatocytes occurs from stage VII through XII of the seminiferous epithelial cycle (see Fig. 2) whereas spermatid labeling is restricted to very few stages. DNA strand breaks in 100% of the nonproliferative elongating spermatids were detected at step 9 (stage IX), where homogeneous nuclear staining was observed (Fig. 1, C and D) and continued throughout subsequent stages X and XI (Fig. 1, E–H). However, during progression from stage IX to stage XI, the number of TUNEL-positive spermatid nuclei declined significantly as chromatin remodeling and condensation proceeded (Fig. 1K). Within seminiferous tubules at stage XI, TUNEL-positive spermatids were still clearly visible along with spermatids showing no TUNEL staining. At stage XII, the homogenous nuclear TUNEL positivity was lost and restricted to a narrow region of the spermatid nucleus (Fig. 1, G and H) whereas at subsequent stages of the cycle (I–VIII), TUNEL positivity was no longer observed in condensing spermatids (Fig. 1, I and J). No TUNEL positivity was observed in the round spermatids also present at these later stages. Moreover, TUNEL assay performed on epididymides cross sections did not reveal DNA strand breaks in epididymal sperm (data not shown). Thus, DNA strand breaks in mouse spermatids is stage-specific and restricted to steps 9–11 (Fig. 1L).



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  		FIG. 1. TUNEL of endogenous DNA strand breaks on adult mouse testis sections at low magnification (A, B) and high magnification (C–J). A) Overlay of TUNEL (green) and DAPI (blue) nuclear staining demonstrating DNA strand breakage according to different epithelium stages. S, Early spermatocytes (arrowhead); ES, elongating spermatids (arrow). B) TUNEL alone. C, D) TUNEL/DAPI and TUNEL staining of stage IX tubule, strong and homogeneous nuclear staining is detected within step 9 elongating spermatids and in spermatocytes. E, F) TUNEL/DAPI and TUNEL staining of stage X–XI seminiferous tubule showing the presence of (both) TUNEL-positive (Es+, arrowheads) and TUNEL-negative elongated spermatids (Es-, arrows) within the same tubule. G, H) Residual TUNEL positivity in elongated spermatids at stage XII. I, J) TUNEL-negative stage VII–VIII tubule. Stages of mouse seminiferous epithelium cycle are designated by roman numerals. Scale bars = 100 µm in A, B; 50 µm in C–J. K) Stage-specific percentage of TUNEL-positive spermatids. Values are means ± SD from visual scoring of at least five tubules per stage. Asterisk denotes statistical significance at P < 0.01. L) Identification of TUNEL-positive spermatids on the mouse stage chart (green box). TUNEL-positive spermatocytes are also boxed (see Fig. 2). Panel L is adapted from Russell et al. (1990) [19]; used with permission of Cache River Science, an imprint of Quick Publishing, LC, 888-PUBLISH, fax 314-993-4485, Cacheriverpress@sbcglobal.net



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  		FIG. 2. Identification of TUNEL-positive spermatocytes. A) Nuclear (DAPI) staining of stage XII seminiferous tubule with identified germ cells. ES, Elongating spermatids; M, meiosis I or II cells; Z, zygotene spermatocytes. B) Corresponding detection of DNA strand breaks staining showing TUNEL positivity restricted to the zygotene spermatocytes (Z+). C, D) DAPI staining and TUNEL of a stage VII–VIII tubule. CS, Condensing spermatids; RT, round spermatids; P, pachytene spermatocyte; PL, preleptotene spermatocytes. TUNEL positivity at these stages is restricted to the preleptotene spermatocytes (PL+). E, F) DAPI staining and TUNEL of a tubule between stage III and VI. All tubules between these stages are TUNEL negative. SG, Spermatogonia. Scale bar = 20 µm

DNA Strand Breaks in Early Spermatocytes

As outlined above, TUNEL positivity was also found in early spermatocytes. This observation was not surprising because of the well-known increase in double strand breaks required to initiate meiotic recombination. The different germ cell types for some stages are well defined and can be easily differentiated within a given stage by their nuclear structure [19]. Figure 2 displays selected tubules at distinct stages of the cycle. TUNEL-positive spermatocytes were observed as early as the preleptotene stage (Fig. 2, C and D) and up to the zygotene to pachytene transition (Fig. 2, A and B). Some early pachytene spermatocytes from stage I tubule also displayed positivity (not shown). All seminiferous tubules identified as between stages II to VI and harboring spermatogonia were all found to be TUNEL-negative (Fig. 2, E and F). Thus, as expected, the TUNEL labeling confirms that DNA strand breakage also takes place in spermatocytes undergoing meiosis (see boxed area in Fig. 1L).

Assessment of DNA Strand Breaks in Decondensed Mature Spermatozoa

Access to the DNA by the terminal transferase at later stages of spermatid maturation may be prevented by the highly compact nature of chromatin that is found in condensed spermatids. To confirm that the DNA strand breaks are indeed repaired before nuclear condensation, epididymal sperm from mice were submitted to in vitro decondensation by exposure to dithiothreitol followed by high salt extraction as described in Material and Methods. As shown in Figure 3 the epididymal sperm remained TUNEL-negative upon induced decondensation confirming that DNA strand breaks are no longer present at this stage.



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  		FIG. 3. TUNEL analysis of decondensed mouse epididymal sperm. A) Mouse spermatozoa were decondensed as described under Material and Methods. TUNEL assay was performed after reduction and extraction of protamines, on sperm preparation. No DNA breaks were detected in decondensed sperm nuclei. B) DNA counterstained with DAPI. Scale bar = 50 µm

Chromatin Remodeling and DNA Strand Breaks

The detection of hyperacetylated histones during mouse spermiogenesis has been associated with active chromatin remodeling in spermatid nuclei. Using a specific antibody against hyperacetylated H4, we seek to establish whether the transient appearance of DNA strand breaks is associated with those steps of active chromatin remodeling. Figure 4, A and B, display the typical strong TUNEL positivity of stage X–XI tubules. When immunolabeled with the anti-AcH4 antibody, these tubules displayed a strong immunoreactivity for the hyperacetylated form of histone H4 (Fig. 4C) whereas a complete absence of H4 immunoreactivity was observed at stages VII–VIII (not shown). Upon superimposition of the TUNEL and immunolabeling, a perfect nuclear colocalization of TUNEL and AcH4 positivity is observed at stages X–XI (Fig. 4D). Higher magnification of stage XI tubule allows demonstrating that those spermatids having completed the repair of their DNA strand breaks at this stage (TUNEL-negative) are no longer immunoreactive for AcH4 (Fig. 4, E and F). At later stages, the TUNEL-negative tubule becomes strongly immunoreactive for protamine 1, suggesting that protamine deposition and further condensation is coincident with the repair of the DNA strand breaks (Fig. 4, G–J). Hence, these results showing a perfect coincidence between the transient appearance of histone acetylation and the presence of DNA strand breaks strongly suggest that the DNA strand breaks are restricted to chromatin remodeling steps and play an essential role in the process.



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  		FIG. 4. Detection of DNA strand breaks and basic nucleoproteins (AcH4 and P1) in mouse seminiferous tubules sections. TUNEL assay and immunodetection were performed successively. A) TUNEL/DAPI staining of a single seminiferous tubule at stages X–XI and (B) corresponding TUNEL staining alone. C) Immunodetection of hyperacetylated histone H4 (AcH4) in the tubule displayed in A and B. D) Merged pictures of TUNEL and immunodetection of AcH4. Upon superimposition of the pictures, a yellow color is generated corresponding to colocalization of immunofluorescence and TUNEL staining. Histone AcH4-positive and TUNEL-positive labeling colocalize in spermatid nuclei at stage X–XI as indicated by arrows. E, F) Higher magnification showing TUNEL and AcH4 immunofluorescence staining of stage XI mouse seminiferous tubule. E) Immunofluorescent localization of AcH4 in spermatid nuclei (red fluorescence). Sections were counterstained for DNA with DAPI (blue fluorescence). Some spermatids immunonegative for AcH4 staining at this stage are indicated by arrows. F) Colocalization of TUNEL staining and AcH4 immunofluorescence. All TUNEL-positive elongating spermatids are immunoreactive for AcH4 (arrowhead) whereas TUNEL-negative spermatids are not immunoreactive. Scale bar = 20 µm. G) TUNEL/DAPI staining of a single seminiferous tubule at stages VII–VIII and (H) corresponding TUNEL staining alone. (I) Immunolocalization of protamine 1 (P1) within condensed spermatids nuclei of the tubule displayed in G and H. J) Merged TUNEL and P1 immunolocalization. Late spermatids are immunoreactive for P1 but are TUNEL negative (arrowheads). Scale bars = 50 µm in all panels except E and F

TUNEL Labeling of Human Testicular Sections

The reported similarity in spermiogenesis among mammals suggests that the transient appearance of DNA strand breaks should also be found in humans. As discussed, this may represent a sensitive process for the integrity of the male gamete and have important implications for the existing assisted reproductive technologies. In humans, the seminiferous epithelium cycle is divided into six stages [20] (Fig. 5E) and the precise steps where chromatin remodeling occurs in the spermatids are not yet known. To these aims, the coincidence of DNA strand breaks and chromatin remodeling has been investigated on human testis cross sections. Seminiferous tubule sections in human often display more than one stage of the epithelium, depending on the sector being examined. Figure 5A displays a typical result obtained following TUNEL and DAPI counterstaining. In the tubule shown, TUNEL positivity is restricted to the round-shaped spermatids of stage III epithelium (boxed area on the right half of the tubule) whereas the boxed area on the left half of the tubule displays epithelium at stages V–VI, where the nuclei of elongated spermatids are TUNEL negative. No TUNEL positivity was observed at other stages (not shown). Interestingly, as for the mouse, TUNEL-positive spermatids were also found to be immunoreactive for AcH4 (Fig. 5, B–D). Thus, chromatin remodeling in human appears to be restricted to TUNEL-positive step 3 spermatids. The transient appearance of DNA strand breaks is therefore widespread among mammals and appears to represent a fundamental component of the chromatin remodeling process taking place in the spermatids.



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  		FIG. 5. TUNEL assay on human testis sections and colocalization with hyperacetylated histone H4. A) TUNEL/DAPI staining at higher magnification. TUNEL positivity is found within early (step 3) spermatids nuclei (right dashed box). Elongated spermatids present at later stages (left dashed box) are TUNEL negative (arrowhead). B–D) Colocalization of TUNEL and AcH4. B) TUNEL-positive spermatids (green fluorescence); (C) immunolocalization of AcH4 (red fluorescence). D) Merged pictures of TUNEL staining and immunodetection of AcH4. Superimposition of both signals produces a yellow color, demonstrating colocalization. Scale bar = 50 µm in A, 20 µm in B, C, and D. E) Stage map of the seminiferous epithelium cycle in human with the TUNEL-positive step 3 spermatid highlighted in green. Panel E is adapted, with permission, from Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press (Lippincott Williams and Wilkins); 1994: 1363–1434 (Figure 1, page 1366)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a sensitive TUNEL assay, we described the stage-dependent pattern of endogenous DNA strand breaks at midspermiogenesis steps and demonstrated for the first time that it is coincident with chromatin remodeling. In the mouse, DNA strand breakage is initiated early in stage IX (step 9 spermatids), coincident with the onset of nuclear reshaping. In both stages IX and X, the fluorescence is maximal and present in close to 100% of the spermatid population. The TUNEL staining covers the entire nucleus surface, indicating that this component of chromatin remodeling is distributed throughout the whole haploid genome. At stage XI, the seminiferous epithelium consistently displays a reduced number of TUNEL-positive spermatids, while only residual TUNEL positivity is detected at stage XII. We further confirmed that no DNA strand breakage is detected in decondensed epididymal sperm. The absence of TUNEL positivity at later stages of the epithelium cycle is therefore not the result of protection by protamine deposition and increased condensation. The global DNA strand breakage therefore precedes the nuclear condensation process in final stages of spermiogenesis. This therefore supports the concept that proper DNA condensation provided by the protamines contributes to the adequate repair of the DNA strand breaks and to preservation of genetic integrity, as suggested in recent reports [3, 23].

The functional significance of DNA strand breaks in elongating spermatids has been discussed recently [8]. One hypothesis involves the requirement for DNA strand breaks to allow the change in DNA topology [2]. The nucleosome withdrawal presumably leaves several unconstrained negative DNA supercoiled in a topological domain and the introduction of nicks in DNA would provide the swivel effect for the complete elimination of the superhelical tension. Topoisomerases could provide the controlled increase in linking number leading to DNA relaxation because they have the dual capacity to both create and seal DNA nicks. Previous reports have presented evidence that topoisomerase II may play a role in this process [10, 24]. However, Cobb et al. demonstrated that TOP2A, the topoisomerase II isoform mainly expressed in the testis, was not present in the mouse elongating spermatids [25]. In addition, topoisomerase II expression has been found to decrease dramatically in the postmeiotic round spermatids [26]. Clearly, the involvement of topoisomerase activities in the transient strand breakage and ligation deserves further investigation.

Due to the sensitivity of the TUNEL assay used in this study, the demonstration was made that not only a fraction but the whole population of elongating spermatids undergoes strand breakage and repair, which is in contrast with previous reports using less sensitive techniques [11, 12, 27]. Although it is well recognized that testicular germ cell apoptosis normally occurs throughout the seminiferous epithelium cycle [28–30], it is, however, clear that the global character of the process by itself rules out that apoptosis represents a possible cause of the global DNA strand breakage that is observed. Not surprisingly, other features of apoptosis, including TUNEL positivity in the entire cells, blebbing of the membrane, or major chromatin abnormalities, were not found in elongating spermatids.

Interestingly, a layer of cells at the basal compartment appears also TUNEL positive in a significant proportion of the tubules. These TUNEL-positive cells found between stages VII and XII were identified as early spermatocytes undergoing meiotic recombination. This observation is not surprising, as previous results from alkaline elution experiments demonstrated that early spermatocytes had elevated levels of endogenous breaks [31]. This is also in agreement with recent findings of Wayne et al. [12]. Their use of a less sensitive TUNEL technique allowed them to demonstrate that a fraction of preleptotene spermatocytes (stages VII–VIII) was preferentially labeled. The fluorescent technique that we have used indicates, however, that a much larger fraction of spermatocytes displays DNA strand breaks. Labeling of spermatocytes extends from stage VII to stage XII, i.e., up to the transition from the zygotene to the pachytene stages so that some stage I tubules may also be labeled. Thus, the TUNEL-positive meiotic cells include preleptotene, leptotene, zygotene, and some early pachytene spermatocytes. The TUNEL positivity of these cells is consistent with the chromatin reorganization that occurs in spermatocytes as well as the presence of DNA double-strand breaks that precede synapsis and extend to the pachytene stage [32]. As the sensitive TUNEL assays used in these experiments do not differentiate between meiotic and apoptotic DNA strand breakage, it is likely that a small subset of the TUNEL-positive spermatocytes are truly apoptotic.

The process of chromatin remodeling in several species is facilitated by histone H4 acetylation [33]. For instance, it has been demonstrated that hyperacetylated nucleosomal core particles are efficiently disassembled by protamines in vitro [34] and that hyperacetylated H4 is clustered within the highly relaxed structure of the spermatid chromatin [35]. Histone H4 becomes highly acetylated during steps 9–12 in the rat [36] and has been recently shown to be acetylated between steps 9 and 11 in the mouse along with the other core histones [37]. Thus, histone acetylation appears to be tightly regulated [37, 38]. Hyperacetylation of histone H4 therefore represents a precise marker of the structural transition where the histone-to-protamine exchange occurs. In the present article, we have shown a perfect nuclear colocalization between the acetylated H4 and TUNEL positivity, indicating that DNA strand breaks occur exactly during the initial steps of chromatin remodeling. This result is therefore in agreement with the hypothesis that a widespread increase in endogenous DNA nicks is required to eliminate the free DNA supercoils during histone exchange, allowing the binding of transition proteins and protamines to the nonsupercoiled DNA typically found in mature spermatids. In addition, a transient decondensed state provided by the global histone hyperacetylation is likely to offer an opportunity for DNA repair complexes (or topoisomerases) to gain access to DNA, which would not be possible at later steps considering the highly compact nature of the lamellar chromatin obtained following protamination.

The orderly sequence of nuclear protein binding to DNA may facilitate the repair of these transient DNA nicks. When expressed in mammalian cells, the transition proteins 1 (TP1) can stimulate the repair of a reporter plasmid harboring single-strand breaks [39] and both the transition proteins [40] and protamines [41] have been shown to enhance the ligation of short DNA fragments in vitro. In agreement with such a role for these basic proteins in vivo, mice with haploinsufficiency for the protamine 2, were recently shown to have a much higher frequency of sperm with damaged DNA as determined by the Comet assay [23]. In addition, mice harboring a double deletion of both the Tnp1 and Tnp2 genes displayed a clear persistence in DNA strand breaks in those spermatids where the condensation process has been clearly altered by the deletions (M. Zhao, personal communication). It is therefore not surprising that, in many instances, an alteration in the condensation state of the sperm head has been correlated with the presence of DNA strand breaks [42–45]. Aside from an alteration in the condensation process, DNA breaks found in mature sperm may result from failure in the postmeiotic DNA repair or from an increase in reactive oxygen species [46]. An alteration in DNA condensation may also offer an opportunity for endonucleases to attack the DNA phosphate backbone due to the lack of proper protection by the nuclear basic proteins [47].

In the present study, we also demonstrated that human early spermatids displayed strong TUNEL positivity. As for the mouse, the colocalization of AcH4 and TUNEL positivity indicated that the increase in endogenous DNA nicks occurs at those steps of nuclear protein exchange and chromatin remodeling in human. The transient increase in DNA strand breaks during chromatin remodeling therefore appears to be widespread among mammals. Hence, the spermiogenesis steps where the endogenous breaks are being created and repaired may represent a sensitive process for the control of DNA integrity, and impediment in the process may have important genetic consequences. In addition, these findings may raise concerns on current ICSI procedures making use of testicular cells. In nonobstructive azoospermia, ICSI appears as the only available treatment; however, the injection of round spermatid (ROSI) has been recently reported to be far less efficient than that of elongated spermatids or mature spermatozoa [17, 18, 48–50]. According to our results, the chromatin remodeling in humans takes place in early round-shaped spermatids of stage III epithelium. Hence, if round spermatids are being used for ICSI procedures, there is a strong possibility that those spermatids harboring the transient DNA strand breaks be selected [51]. We suggest that the presence of a high number of DNA strand breaks in early spermatids is likely to be responsible for the development failure observed so far after ROSI and represents a potential risk should any embryo be allowed to develop after fertilization with these cells. In support of this hypothesis, sperm DNA strand breaks induced by gamma radiation were found to severely decrease the rate of embryonic development in the mouse after ICSI [52]. Accordingly, we are currently investigating whether the endogenous level of DNA strand breaks in mouse spermatids (steps 9–10) is beyond the repair capacity of the oocyte. In the mouse, ICSI has been successfully achieved using round spermatids [53, 54]. In accordance with the results presented in this article, one likely explanation would be that, in the mouse, DNA strand breakage is restricted to the elongating spermatids but not found in the round spermatids, where chromatin remodeling has not yet proceeded.

In summary, the results presented in this article demonstrate that, in mammals, the chromatin remodeling involves a transient but global appearance of DNA strand breaks. Although the nature of DNA strand breaks and the molecular mechanism involved deserves further investigations, it is clear that this may represent a sensitive step in the control of the genetic integrity of the male gamete. Hence, this may provide important clues for the etiology of sperm DNA fragmentation in subfertile males, e.g., should any persistence in this transient process occurs. In addition, these results may raise a better awareness regarding the genetic risk of ROSI in humans.


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Marvin Meistrich for valuable comments on the manuscript and help with the staging of seminiferous tubules and Remi-Martin Laberge for insightful discussions during the preparation of this manuscript.


   FOOTNOTES
 
1 Supported by grants from the Natural Science and Engineering Council of Canada (grant 155182-99) and from the Canadian Institute of Health Research (grant MOP-37881) to G.B. Back

2 Correspondence: Guylain Boissonneault, Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12ième Ave Nord, Sherbrooke, Québec, Canada J1H 5N4. FAX: 819 564 5340; guylain.boissonneault{at}usherbrooke.ca Back

Received: 25 August 2003.

First decision: 12 September 2003.

Accepted: 14 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ward WS. The structure of the sleeping genome: implications of sperm DNA organization of somatic cells. J Cell Biochem 1994 55:77-82[CrossRef][Medline]
  2. Risley MS, Einheber S, Bumcrot DA. Changes in DNA topology during spermatogenesis. Chromosoma 1986 94:217-227[CrossRef][Medline]
  3. Cho C, Willis WD, Goulding EH, Haesook JH, Choi YC, Hecht NB, Eddy EM. Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 2001 28:82-86[CrossRef][Medline]
  4. Zhao M, Shirley CR, Yu YE, Mohapatra B, Zhang Y, Unni E, Deng JM, Arango NA, Terry NHA, Weil MM, Russell LD, Behringer RR, Meistrich ML. Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice. Mol Cell Biol 2001 21:7243-7255[Abstract/Free Full Text]
  5. Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, Weil MM, Behringer RR, Meistrich ML. Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice. Proc Natl Acad Sci U S A 2000 97:4683-4688[Abstract/Free Full Text]
  6. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P, Claussen OP. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999 14:1039-1049[Abstract/Free Full Text]
  7. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002 23:25-43[Medline]
  8. Boissonneault G. Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair. FEBS Lett 2002 514:111-114[CrossRef][Medline]
  9. McPherson SM, Longo FJ. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 1992 31:268-279[CrossRef][Medline]
  10. McPherson SM, Longo FJ. Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II. Dev Biol 1993 158:122-130[CrossRef][Medline]
  11. Sakkas D, Manicardi G, Bianchi PG, Bizzaro D, Bianchi U. Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod 1995 52:1149-1155[Abstract]
  12. Wayne CM, Sutton K, Wilkinson MF. Expression of the pem homeobox gene in Sertoli cells increases the frequency of adjacent germ cells with deoxyribonucleic acid strand breaks. Endocrinology 2002 143:4875-4885[Abstract/Free Full Text]
  13. Smith A, Haaf T. DNA nicks and increased sensitivity of DNA to fluorescence in situ end labeling during functional spermiogenesis. Biotechniques 1998 25:496-502[Medline]
  14. Dadoune JP. The nuclear status of human sperm cells. Micron 1995 26:323-345
  15. Wouters-Tyrou D, Martinage A, Chevaillier P, Sautiere P. Nuclear basic proteins in spermiogenesis. Biochimie 1998 80:117-128[Medline]
  16. Ghazzawi IM, Alhasani S, Taher M, Souso S. Reproductive capacity of round spermatids compared with mature spermatozoa in a population of azoospermic men. Hum Reprod 1999 14:736-740[Abstract/Free Full Text]
  17. Vicdan K, Isik AZ, Deliliase L. Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure. J Assist Reprod Genet 2001 18:78-86[CrossRef][Medline]
  18. Urman B, Alatas C, Aksoy S, Mercan R, Nuhoglu A, Mumcu A, Isiklar A, Balaban B. Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection. Hum Reprod 2002 17:741-743[Abstract/Free Full Text]
  19. Russell LD, Ettlin RA, Hikim AP, Clegg E. Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River Press; 1990
  20. Clermont Y. The cycle of the seminiferous epithelium in man. Am J Anat 1963 112:35-51[CrossRef][Medline]
  21. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997 56:602-607[Abstract]
  22. Szczygiel MA, Ward WS. Combination of dithiothreitol and detergent treatment of spermatozoa causes paternal chromosomal damage. Biol Reprod 2002 67:1532-1537[Abstract/Free Full Text]
  23. Cho C, Jung-Ha H, Willis WD, Goulding EH, Stein P, Xu Z, Schultz RM, Hecht NB, Eddy EM. Protamine-2 deficiency leads to sperm DNA damage and embryo death in mice. Biol Reprod 2003 69:211-217[Abstract/Free Full Text]
  24. Chen JL, Longo FJ. Expression and localization of DNA topoisomerase II during rat spermatogenesis. Mol Reprod Dev 1996 45:93-105[CrossRef][Medline]
  25. Cobb J, Miyaike M, Kikuchi A, Handel MA. Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase II alpha localization and chromosome condensation. Chromosoma 1999 108:412-425[CrossRef][Medline]
  26. Cobb J, Reddy RK, Park C, Handel MA. Analysis of expression and function of topoisomerase I and II during meiosis in male mice. Mol Reprod Dev 1997 46:489-498[CrossRef][Medline]
  27. Oldereid NB, Angelis PD, Wiger R, Clausen OP. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. Mol Hum Reprod 2001 7:403-408[Abstract/Free Full Text]
  28. Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. Embo J 1997 16:2262-2270[CrossRef][Medline]
  29. Furuchi T, Masuko K, Nishimune Y, Obinata M, Matsui Y. Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development 1996 122:1703-1709[Abstract]
  30. Koji T, Hishikawa Y. Germ cell apoptosis and its molecular trigger in mouse testes. Arch Histol Cytol 2003 66:1-16[CrossRef][Medline]
  31. Joshi DS, Yick J, Murray D, Meistrich ML. Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells. Radiat Res 1990 121:274-281[Medline]
  32. Mahadevaiah SK, Turner JM, Baudat F, Rogakou EP, de Boer P, Blanco-Rodriguez J, Jasin M, Keeney S, Bonner WM, Burgoyne PS. Recombinational DNA double-strand breaks in mice precede synapsis (comment). Nat Genet 2001 27:271-276[CrossRef][Medline]
  33. Grimes SR Jr, Henderson N. Hyperacetylation of histone H4 in rat testis spermatids. Exp Cell Res 1984 152:91-97[CrossRef][Medline]
  34. Oliva R, Bazett-Jones D, Mezquita C, Dixon GH. Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins. J Biol Chem 1987 262:17016-17025[Abstract/Free Full Text]
  35. Christensen ME, Rattner JB, Dixon GH. Hyperacetylation of histone H4 promotes chromatin decondensation before histone replacement by protamines during spermatogenesis in rainbow trout. Nucleic Acids Res 1984 12:4575-4592[Abstract/Free Full Text]
  36. Meistrich ML, Trostle-Weige PK, Lin R, Bhatnagar YM, Allis CD. Highly acetylated H4 is associated with histone displacement in rat spermatids. Mol Reprod Dev 1992 31:170-181[CrossRef][Medline]
  37. Hazzouri M, Pivot-Pajot C, Faure AK, Usson Y, Pelletier R, Sele B, Khochbin S, Rousseaux S. Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases. Eur J Cell Biol 2000 79:950-960[CrossRef][Medline]
  38. Lahn BT, Tang ZL, Zhou J, Barndt RJ, Parvinen M, Allis CD, Page DC. Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis. Proc Natl Acad Sci U S A 2002 99:8707-8712[Abstract/Free Full Text]
  39. Caron N, Veilleux S, Boissonneault G. Stimulation of DNA repair by the spermatidal TP1 protein. Mol Reprod Dev 2001 58:437-443[CrossRef][Medline]
  40. Lévesque D, Veilleux S, Caron N, Boissonneault G. Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2. Biochem Biophys Res Commun 1998 252:602-609[CrossRef][Medline]
  41. Sheflin LG, Fucile NW, Spaulding SW. HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: phosphorylation of HMG 14 at ser 20 specifically inhibit intermolecular ligation. Biochem Biophys Res Commun 1991 174:660-666[CrossRef][Medline]
  42. Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res 1993 207:202-205[CrossRef][Medline]
  43. Manicardi GC, Tombacco A, Bizzaro D, Bianchi U, Bianchi PG, Sakkas D. DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays. Histochem J 1998 30:33-39[CrossRef][Medline]
  44. Aravindan GR, Moudgal NR. Susceptibility of sperm chromatin to acid denaturation in situ: a study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata). Arch Androl 1998 40:29-41[Medline]
  45. Bianchi PG, Manicardi GC, Bizzaro D, Bianchi U, Sakkas D. Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biol Reprod 1993 49:1083-1088[Abstract]
  46. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998 59:1037-1046[Abstract/Free Full Text]
  47. Meistrich ML, Mohapatra B, Shirley CR, Zhao M. Roles of transition nuclear proteins in spermiogenesis. Chromosoma 2003 111:483-488[Medline]
  48. Khalili MA, Aflatoonian A, Zavos PM. Intracytoplasmic injection using spermatids and subsequent pregnancies: Round versus elongated spermatids. J Assist Reprod Genet 2002 19:84-86[CrossRef][Medline]
  49. Kahraman S, Polat G, Samli M, Sozen E, Ozgun OD, Dirican K, Ozbicer T. Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum Reprod 1998 13:104-110[Abstract/Free Full Text]
  50. Schoysman R, Vanderzwalmen P, Bertin G, Nijs M, Van Damme B. Oocyte insemination with spermatozoa precursors. Curr Opin Urol 1999 9:541-545[CrossRef][Medline]
  51. Johnson L, Neaves WB, Barnard JJ, Keillor GE, Brown SW, Yanagimachi R. A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules. Biol Reprod 1999 61:927-934[Abstract/Free Full Text]
  52. Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999 284:696-704[CrossRef][Medline]
  53. Marh J, Tres LL, Yamazaki Y, Yanagimachi R, Kierszenbaum AL. Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo developed mature oocytes. Biol Reprod 2003 69:169-176[Abstract/Free Full Text]
  54. Hayashi S, Yang J, Christenson L, Yanagimachi R, Hecht NB. Mouse preimplantation embryos developed from oocytes injected with round spermatids or spermatozoa have similar but distinct patterns of early mRNA expression. Biol Reprod 2003 69:1170-1176[Abstract/Free Full Text]



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