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Nature and origin of transient DNA strand breaks during spermiogenesis
International Congress Series 1271 (2004) 189 – 192
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Nature and origin of transient DNA strand breaks during spermiogenesis Re´mi-Martin Laberge, Guylain Boissonneault * De´partement de Biochimie, Faculte´ de Me´decine, Biochimie, Universite´ de Sherbrooke, 3001, 12e Avenue nord, Sherbrooke, Quebec, Canada J1H 5N4
Abstract. During mid-spermiogenesis steps, transient DNA strand breaks appear in the whole population of elongating spermatids. This would be necessary to relieve the free DNA supercoils remaining after histones displacement since the DNA is no longer supercoiled in the mature spermatozoa. Histones hyperacetylation is coincident with the chromatin remodelling steps. This post-translational modification appears to be essential for histones displacement and is found to colocalize with the transient DNA strand breaks suggesting that it may represent a prerequisite to allow strand breakages to form. The negative supercoils resulting from DNA wrapping around the nucleosome could be eliminated by the enzymatic action of topoisomerases known to relax negative supercoils. The transition proteins (TPs) and protamines, which sequentially bind DNA after histones removal, could provide the appropriate scaffold to support the final resealing steps of the process. We hypothesize that the DNA strand breakage and ligation may represent sensitive steps with the potential to alter fertility if the sequence of event is altered. The mutagenic potential of these events may also result in fertility and/or developmental problems. D 2004 Elsevier B.V. All rights reserved. Keywords: Spermiogenesis; DNA strand break; Histone acetylation; Topoisomerase; DNA condensation
1. Introduction The presence of DNA strand breaks in mature spermatozoa has been strongly correlated with impaired fertility [1]. Unrepaired double- or single-stranded breakage has mutagenic potential and may represent a genetic threat to the embryo. The mechanism by which those breaks are created in spermatozoa remains unknown, but some evidence from the recent literature indicate that impaired chromatin integrity is correlated with DNA strand breaks in the mature sperm [2]. Since the sperm chromatin structure is determined solely at midspermiogenesis steps, the etiology of DNA strand breaks may lie within this important phase of the male gamete maturation. The recent demonstration by our group that chromatin remodelling involves the formation of transient DNA strand breaks in the whole population of spermatids suggests that impairment in that process could result in the
* Corresponding author. Tel.: +1-819-564-5443; fax: +1-819-564-5340. E-mail address: [email protected] (G. Boissonneault). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.05.082
190
R.-M. Laberge, G. Boissonneault / International Congress Series 1271 (2004) 189–192
persistence of DNA strand breaks in spermatozoa [3]. We surmise that understanding the mechanism by which DNA strand breaks are created and repaired will lead to important clues for idiopathic cases of male infertility. 2. Histones hyperacetylation During spermatid elongation, the nucleosome core is replaced by other DNA-condensing proteins. In mammals, transition proteins (TPs) initiate the condensation process which is completed by the deposition of protamines. Post-translational modifications, particularly acetylation, are thought to facilitate the process [3 –5]. Interestingly, a decrease in histone acetylation in elongating spermatid has been associated with a reduction of fertility in human [4]. Histone hyperacetylation at these steps is probably created by an unbalance in the histone acetyl transferase/histone deacetylase (HAT/HDAC) ratio since in vitro treatment of round spermatid with trichostatin A (TSA), an HDAC inhibitor, leads to hyperacetylation of H4 [5]. Histone N-termini usually bind DNA and have a marked influence of the different levels of chromatin organization. Underacetylated chromatin is less accessible for the DNA transactions. Round spermatids usually display no DNA strand breaks. We demonstrated that, when treated with TSA, histone hyperacetylation occurs and DNA strand breakage is detected. Therefore, histone acetylation is somehow involved in the generation of DNA strand breaks. This also implies that the DNA strand breakage activity is already present in round spermatids but does not gain access to DNA in the normal acetylation state of these cells (unpublished results). 3. Elimination of supercoiling by DNA strand breaks DNA wrapping around the histone core generates constrained negative supercoils. Supercoils are no longer found in mature spermatozoa. Once the nucleosomes are withdrawn, the torsional stress, in the form of free DNA supercoils must be relieved which should require the formation of either single- or double-stranded breaks. Such topological activities are usually performed by topoisomerases able to change the DNA linking number in steps of one (topo I) or two (topo II) in order to relax DNA. As outlined above, we have shown that DNA strand breaks appear in the whole population of elongating spermatids lending support to this concept [3]. Ten years ago, it has been proposed that topoisomerase II may perform such a task during the chromatin remodelling steps in the rat [6]. The use of a topo II inhibitor (suramin) in short-term culture of mouse elongating spermatids can prevent the formation of strand breakage (unpublished results). In addition, preliminary Comet assays performed in either alkaline or neutral conditions indicate that a large proportion of DNA double-stranded breaks are formed. Taken together, these results are in accordance with the hypothesis that the activity of a type II topoisomerase is present in elongating spermatids. However, at this point, the possibility that strand breakage and ligation are performed by different enzymes (uncoupling) may not be excluded. In any case, the DNA strand breakage and ligation may be error-prone and offer an opportunity for mutations. A somatic cell type repair mechanism appears unlikely considering that nearly all DNA transactions are abolished in the condensing spermatids. However, a trinucleotide expansion repair mechanism has been shown to operate in late spermatids and spermatozoa [7]. This ultimate repair process could be the
R.-M. Laberge, G. Boissonneault / International Congress Series 1271 (2004) 189–192
191
source of paternally transmitted hereditary disease [8]. This emphasizes the requirement for a reliable mechanism to remove the superhelical tension without altering the free DNA ends. 4. DNA condensation The proper packaging of the paternal chromosome is essential, and infertility may arise if alterations in the process occur. Following (or during) histone withdrawal, the major mammalian transition proteins, TP1 and TP2, apparently initiate the process around steps 10 –12 of spermiogenesis. The role of the transition proteins is not well understood, but it is clear that they perform an essential role in the chromatin remodelling sequence since mice with deletion of both TPs are no longer fertile and severe alterations of the nuclear events of spermiogenesis are observed. The architectural DNA-binding properties of TPs and protamines may provide the proper scaffolding to help the DNA strand break repair process as suggested by in vitro experiments [9,10]. The protamines also appear to be essential to the DNA integrity as mice with haploinsufficiency in protamine are infertile and chromatin integrity is altered [11]. DNA strand breaks are associated with alteration in sperm DNA condensation [2]. In addition, a decrease of histone acetylation in elongating spermatids is associated with alteration in the histones-to-protamines exchange and infertility [4]. Collectively, these data support the concept that the sperm DNA packaging process, taking place in the spermatids, is closely linked to DNA integrity. 5. Discussion From the above, the following scheme or sequence of event emerges (Fig. 1): Histone acetylation during spermiogenesis is required in order to promote DNA accessibility allowing the DNA strand breakage and ligation to proceed using the activity of a
Fig. 1. Model of sequential removal of torsional stress during spermiogenesis. (1) Spermatids 1 – 8, DNA is wrapped around nucleosomes, the histones tails are constraining DNA on nucleosomes. (2) Spermatids 8 – 9, HAT/HDAC ratio rises, the histones tails become hyperacetylated and DNA become accessible. (3) Spermatids 9 – 10, chromatin relaxing continues while a nuclease (possibly topoisomerase II) makes DNA strand breaks. (4) Spermatids 10 – 11, topo II activity continues and nucleosomes are removed. (5) Spermatids 12 – 13, DNA strand breaks are repaired after the removal of the negative supercoils allowing the transition proteins binding to DNA.
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R.-M. Laberge, G. Boissonneault / International Congress Series 1271 (2004) 189–192
topological enzyme (topoisomerase II?). Histone acetylation also facilitates their own withdrawal from the DNA. The transient DNA double-strand breaks can now be produced to remove torsional stress in DNA. The TPs and protamines can now bind DNA and help maintain the non-supercoiled state [12]. The binding of these major DNA condensing proteins would, in turn, stimulate the repair of the DNA strand breaks. If a perturbation occurs in one of these processes, this will likely result in the persistence of DNA damages and/or inappropriate condensation state leading to a decrease of the fertilizing potential of the male gamete. As more investigations are performed, the transient DNA strand breakage at midspermiogenesis steps may prove to represent a sensitive step for the genetic integrity of the male gamete. The sensitive nature of this mechanism may be at the origin of DNA strand breaks found in the semen of subfertile males. References [1] E. Host, et al., DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility, Acta Obstet. Gynecol. Scand. 78 (7) (1999 Aug.) 622 – 625. [2] G.C. Manicardi, et al., DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays, Histochem. J. 30 (1) (1998 Jan.) 33 – 39. [3] L. Marcon, G. Boissonneault, Transient DNA strand breaks during mouse and human spermiogenesis: new insights in stage specificity and link to chromatin remodeling, Biol. Reprod. 70 (4) (2004 Apr.) 910 – 918. [4] V. Sonnack, et al., Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis, Andrologia 34 (6) (2002 Dec.) 384 – 390. [5] M. Hazzouri, et al., Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases, Eur. J. Cell Biol. 79 (12) (2000 Dec.) 950 – 960. [6] S.M. McPherson, F.J. Longo, Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II, Dev. Biol. 158 (1) (1993 Jul.) 122 – 130. [7] C.T. McMurray, I.V. Kortun, Repair in haploid male germ cells occurs late in differentiation as chromatin is condensing, Chromosoma 111 (8) (2003 May) 505 – 508. [8] C.T. McMurray, DNA secondary structure: a common and causative factor for expansion in human disease, Proc. Natl. Acad. Sci. U. S. A. 96 (5) (1999 Mar. 2) 1823 – 1825 (Review). [9] N. Caron, S. Veilleux, G. Boissonneault, Stimulation of DNA repair by the spermatidal TP1 protein, Mol. Reprod. Dev. 58 (4) (2001 Apr.) 437 – 443. [10] G. Boissonneault, Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair, FEBS Lett. 514 (2 – 3) (2002 Mar. 13) 111 – 114. [11] C. Cho, et al., Haploinsufficiency of protamine-1 or -2 causes infertility in mice, Nat Genet. 28 (1) (2001 May) 82 – 86. [12] D. Levesque, et al., Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2, Biochem. Biophys. Res. Commun. 252 (3) (1998 Nov. 27) 602 – 609.
www.ics-elsevier.com
Nature and origin of transient DNA strand breaks during spermiogenesis Re´mi-Martin Laberge, Guylain Boissonneault * De´partement de Biochimie, Faculte´ de Me´decine, Biochimie, Universite´ de Sherbrooke, 3001, 12e Avenue nord, Sherbrooke, Quebec, Canada J1H 5N4
Abstract. During mid-spermiogenesis steps, transient DNA strand breaks appear in the whole population of elongating spermatids. This would be necessary to relieve the free DNA supercoils remaining after histones displacement since the DNA is no longer supercoiled in the mature spermatozoa. Histones hyperacetylation is coincident with the chromatin remodelling steps. This post-translational modification appears to be essential for histones displacement and is found to colocalize with the transient DNA strand breaks suggesting that it may represent a prerequisite to allow strand breakages to form. The negative supercoils resulting from DNA wrapping around the nucleosome could be eliminated by the enzymatic action of topoisomerases known to relax negative supercoils. The transition proteins (TPs) and protamines, which sequentially bind DNA after histones removal, could provide the appropriate scaffold to support the final resealing steps of the process. We hypothesize that the DNA strand breakage and ligation may represent sensitive steps with the potential to alter fertility if the sequence of event is altered. The mutagenic potential of these events may also result in fertility and/or developmental problems. D 2004 Elsevier B.V. All rights reserved. Keywords: Spermiogenesis; DNA strand break; Histone acetylation; Topoisomerase; DNA condensation
1. Introduction The presence of DNA strand breaks in mature spermatozoa has been strongly correlated with impaired fertility [1]. Unrepaired double- or single-stranded breakage has mutagenic potential and may represent a genetic threat to the embryo. The mechanism by which those breaks are created in spermatozoa remains unknown, but some evidence from the recent literature indicate that impaired chromatin integrity is correlated with DNA strand breaks in the mature sperm [2]. Since the sperm chromatin structure is determined solely at midspermiogenesis steps, the etiology of DNA strand breaks may lie within this important phase of the male gamete maturation. The recent demonstration by our group that chromatin remodelling involves the formation of transient DNA strand breaks in the whole population of spermatids suggests that impairment in that process could result in the
* Corresponding author. Tel.: +1-819-564-5443; fax: +1-819-564-5340. E-mail address: [email protected] (G. Boissonneault). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.05.082
190
R.-M. Laberge, G. Boissonneault / International Congress Series 1271 (2004) 189–192
persistence of DNA strand breaks in spermatozoa [3]. We surmise that understanding the mechanism by which DNA strand breaks are created and repaired will lead to important clues for idiopathic cases of male infertility. 2. Histones hyperacetylation During spermatid elongation, the nucleosome core is replaced by other DNA-condensing proteins. In mammals, transition proteins (TPs) initiate the condensation process which is completed by the deposition of protamines. Post-translational modifications, particularly acetylation, are thought to facilitate the process [3 –5]. Interestingly, a decrease in histone acetylation in elongating spermatid has been associated with a reduction of fertility in human [4]. Histone hyperacetylation at these steps is probably created by an unbalance in the histone acetyl transferase/histone deacetylase (HAT/HDAC) ratio since in vitro treatment of round spermatid with trichostatin A (TSA), an HDAC inhibitor, leads to hyperacetylation of H4 [5]. Histone N-termini usually bind DNA and have a marked influence of the different levels of chromatin organization. Underacetylated chromatin is less accessible for the DNA transactions. Round spermatids usually display no DNA strand breaks. We demonstrated that, when treated with TSA, histone hyperacetylation occurs and DNA strand breakage is detected. Therefore, histone acetylation is somehow involved in the generation of DNA strand breaks. This also implies that the DNA strand breakage activity is already present in round spermatids but does not gain access to DNA in the normal acetylation state of these cells (unpublished results). 3. Elimination of supercoiling by DNA strand breaks DNA wrapping around the histone core generates constrained negative supercoils. Supercoils are no longer found in mature spermatozoa. Once the nucleosomes are withdrawn, the torsional stress, in the form of free DNA supercoils must be relieved which should require the formation of either single- or double-stranded breaks. Such topological activities are usually performed by topoisomerases able to change the DNA linking number in steps of one (topo I) or two (topo II) in order to relax DNA. As outlined above, we have shown that DNA strand breaks appear in the whole population of elongating spermatids lending support to this concept [3]. Ten years ago, it has been proposed that topoisomerase II may perform such a task during the chromatin remodelling steps in the rat [6]. The use of a topo II inhibitor (suramin) in short-term culture of mouse elongating spermatids can prevent the formation of strand breakage (unpublished results). In addition, preliminary Comet assays performed in either alkaline or neutral conditions indicate that a large proportion of DNA double-stranded breaks are formed. Taken together, these results are in accordance with the hypothesis that the activity of a type II topoisomerase is present in elongating spermatids. However, at this point, the possibility that strand breakage and ligation are performed by different enzymes (uncoupling) may not be excluded. In any case, the DNA strand breakage and ligation may be error-prone and offer an opportunity for mutations. A somatic cell type repair mechanism appears unlikely considering that nearly all DNA transactions are abolished in the condensing spermatids. However, a trinucleotide expansion repair mechanism has been shown to operate in late spermatids and spermatozoa [7]. This ultimate repair process could be the
R.-M. Laberge, G. Boissonneault / International Congress Series 1271 (2004) 189–192
191
source of paternally transmitted hereditary disease [8]. This emphasizes the requirement for a reliable mechanism to remove the superhelical tension without altering the free DNA ends. 4. DNA condensation The proper packaging of the paternal chromosome is essential, and infertility may arise if alterations in the process occur. Following (or during) histone withdrawal, the major mammalian transition proteins, TP1 and TP2, apparently initiate the process around steps 10 –12 of spermiogenesis. The role of the transition proteins is not well understood, but it is clear that they perform an essential role in the chromatin remodelling sequence since mice with deletion of both TPs are no longer fertile and severe alterations of the nuclear events of spermiogenesis are observed. The architectural DNA-binding properties of TPs and protamines may provide the proper scaffolding to help the DNA strand break repair process as suggested by in vitro experiments [9,10]. The protamines also appear to be essential to the DNA integrity as mice with haploinsufficiency in protamine are infertile and chromatin integrity is altered [11]. DNA strand breaks are associated with alteration in sperm DNA condensation [2]. In addition, a decrease of histone acetylation in elongating spermatids is associated with alteration in the histones-to-protamines exchange and infertility [4]. Collectively, these data support the concept that the sperm DNA packaging process, taking place in the spermatids, is closely linked to DNA integrity. 5. Discussion From the above, the following scheme or sequence of event emerges (Fig. 1): Histone acetylation during spermiogenesis is required in order to promote DNA accessibility allowing the DNA strand breakage and ligation to proceed using the activity of a
Fig. 1. Model of sequential removal of torsional stress during spermiogenesis. (1) Spermatids 1 – 8, DNA is wrapped around nucleosomes, the histones tails are constraining DNA on nucleosomes. (2) Spermatids 8 – 9, HAT/HDAC ratio rises, the histones tails become hyperacetylated and DNA become accessible. (3) Spermatids 9 – 10, chromatin relaxing continues while a nuclease (possibly topoisomerase II) makes DNA strand breaks. (4) Spermatids 10 – 11, topo II activity continues and nucleosomes are removed. (5) Spermatids 12 – 13, DNA strand breaks are repaired after the removal of the negative supercoils allowing the transition proteins binding to DNA.
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R.-M. Laberge, G. Boissonneault / International Congress Series 1271 (2004) 189–192
topological enzyme (topoisomerase II?). Histone acetylation also facilitates their own withdrawal from the DNA. The transient DNA double-strand breaks can now be produced to remove torsional stress in DNA. The TPs and protamines can now bind DNA and help maintain the non-supercoiled state [12]. The binding of these major DNA condensing proteins would, in turn, stimulate the repair of the DNA strand breaks. If a perturbation occurs in one of these processes, this will likely result in the persistence of DNA damages and/or inappropriate condensation state leading to a decrease of the fertilizing potential of the male gamete. As more investigations are performed, the transient DNA strand breakage at midspermiogenesis steps may prove to represent a sensitive step for the genetic integrity of the male gamete. The sensitive nature of this mechanism may be at the origin of DNA strand breaks found in the semen of subfertile males. References [1] E. Host, et al., DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility, Acta Obstet. Gynecol. Scand. 78 (7) (1999 Aug.) 622 – 625. [2] G.C. Manicardi, et al., DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays, Histochem. J. 30 (1) (1998 Jan.) 33 – 39. [3] L. Marcon, G. Boissonneault, Transient DNA strand breaks during mouse and human spermiogenesis: new insights in stage specificity and link to chromatin remodeling, Biol. Reprod. 70 (4) (2004 Apr.) 910 – 918. [4] V. Sonnack, et al., Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis, Andrologia 34 (6) (2002 Dec.) 384 – 390. [5] M. Hazzouri, et al., Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases, Eur. J. Cell Biol. 79 (12) (2000 Dec.) 950 – 960. [6] S.M. McPherson, F.J. Longo, Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II, Dev. Biol. 158 (1) (1993 Jul.) 122 – 130. [7] C.T. McMurray, I.V. Kortun, Repair in haploid male germ cells occurs late in differentiation as chromatin is condensing, Chromosoma 111 (8) (2003 May) 505 – 508. [8] C.T. McMurray, DNA secondary structure: a common and causative factor for expansion in human disease, Proc. Natl. Acad. Sci. U. S. A. 96 (5) (1999 Mar. 2) 1823 – 1825 (Review). [9] N. Caron, S. Veilleux, G. Boissonneault, Stimulation of DNA repair by the spermatidal TP1 protein, Mol. Reprod. Dev. 58 (4) (2001 Apr.) 437 – 443. [10] G. Boissonneault, Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair, FEBS Lett. 514 (2 – 3) (2002 Mar. 13) 111 – 114. [11] C. Cho, et al., Haploinsufficiency of protamine-1 or -2 causes infertility in mice, Nat Genet. 28 (1) (2001 May) 82 – 86. [12] D. Levesque, et al., Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2, Biochem. Biophys. Res. Commun. 252 (3) (1998 Nov. 27) 602 – 609.