ORIGINAL ARTICLE
In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation 1, A. Caille1, C. Zumoffen1, S. Ghersevich1, L. Bahamondes2 & M. J. Munuce1 J. Cicare 1 Laboratory of Reproductive Studies, Clinical Biochemistry Area, School of Biochemical and Pharmaceutical Sciences, National University of Rosario, Rosario, Argentina; 2 Human Reproduction Unit, Department of Obstetrics and Gynecology, School of Medical Sciences, University of Campinas (UNICAMP) and National Institute of Hormones and Women’s Health, Campinas, SP, Brazil
Keywords DNA fragmentation—dynamics—SCD test— sperm Correspondence Marıa Jos e Munuce, Laboratory of Reproductive Studies, Biochemical Chemistry Area, School of Biochemical and Pharmaceutical Sciences, University of Rosario, Suipacha 531, 2000 Rosario, Argentina. Tel.: + 54 341 4804597; Fax: + 54 341 4804592; E-mail: [email protected] Accepted: July 08, 2014 doi: 10.1111/and.12337
Summary The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham0 s F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.
Introduction Because of their aerobic metabolism, spermatozoa in culture generate low levels of reactive oxygen species (ROS), which include superoxide anion, hydrogen peroxide, hydroperoxyl radicals and hydroxyl radicals (Aitken, 1995). Many beneficial effects of ROS have been described, including their ability to stimulate hyperactivated motility and tyrosine phosphorylation in several proteins associated with capacitation (de Lamirande et al., 1997). However, high levels of ROS generated by infiltrating leucocytes or abnormal spermatozoa can overwhelm seminal plasma antioxidant defences, leading to the peroxidation of plasma membrane polyunsaturated fatty acids (LP) and DNA damage (Aitken & Clarkson, 1987). It is widely accepted that the presence of damaged DNA in mature ejaculated spermatozoa is associated with a range of adverse clinical outcomes including infertility, miscarriage and diseases in offspring (Agarwal & Said, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
2003; Collins et al., 2008). Moreover, a recent study reported on the ‘dynamics of sperm DNA fragmentation’ during in vitro incubation time, which was associated with a reduction in the fertilising potential (Gosalvez et al., 2011a). Considering that in most protocols of assisted reproductive technology (ART), selected human spermatozoa may remain incubated in vitro for several hours awaiting fertilisation (Liu et al., 2011). The aim of this study was to investigate the oxidative process associated with sperm incubation under capacitating conditions and its impact on DNA fragmentation and sperm function. Materials and methods The study was approved by the Institutional Review Board of the School of Biochemical and Pharmaceutical Sciences of the National University of Rosario, Argentina. Written informed consent that complies with all the 1
J. Cicare et al.
Dynamic of DNA fragmentation
Declaration of Helsinki Principles was signed by all the semen donors (n = 26). Semen sample and sperm processing Semen samples were obtained from healthy normozoospermic donors according to the World Health Organization guidelines (WHO, 2010). Only samples with 80% of viable cells. The ability to undergo the hFF-induced AR was also measured. Results showed that spontaneous and induced AR values were comparable in the control and H2O2-treated group, respectively: basal: 13.8 0.9% vs. induced AR: 40.6 4.6% and basal: 13.9 1.5% vs. induced AR: 37.8 5.4% (P < 0.001; n = 5). Furthermore, no differences were found in the TyrP pattern, either in the control or treatment groups, suggesting that capacitation had taken place during in vitro incubation in both situations (Fig. 4; NS; n = 5). Discussion
Fig. 1 Kinetics of redox activity (ReA) during sperm incubation under capacitating conditions. ReA was determined at 3, 6 and 22 h using the chemiluminescent probe luminol. Results are expressed as mean SEM, (n = 16). *(P < 0.05) when compared to semen and other incubation times.
Fig. 2 Kinetics of lipid peroxidation (LP) during sperm incubation under capacitating conditions. LP was measured using the thiobarbituric acid assay. Results are expressed as mean SEM, (n = 16). *(P < 0.05) when compared to semen and other incubation times.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
During sperm preparation for ART, gametes must be cocultured for several hours. Some forms of iatrogenic oxidative stress associated with the method used to prepare spermatozoa may induce DNA damage (Twigg et al., 1998a). The principal objective of the present study was to determine the kinetics of ReA and LP on spermatozoa in culture. No significant variation in ReA or LP levels was detected after 3 or 6 h of sperm incubation under optimal conditions. It is believed that the bovine serum albumin used as a cholesterol acceptor for capacitation
Fig. 3 Effect on DNA status during sperm incubation under capacitaing conditions and acrosome reaction. DNA was analysed using the sperm chromatin dispersion test. Results are expressed as mean SEM (n = 5). a,bP < 0.05 vs. control group, cP < 0.001 vs. post-swim-up, d,eP < 0.001 vs. post-swim-up and post-capacitation.
3
J. Cicare et al.
Dynamic of DNA fragmentation
(a)
(b)
(c)
Fig. 4 Analysis of phosphorylation on tyrosine residues. Panel (a) Proteins transferred to the blot (stained with Ponceau red) in the control (C) and treated groups (T). Panel (b) Immunodetection of TyrP using a monoclonal antiphosphotyrosine antibody. Molecular weight standards (MW) are indicated on the left side of Panel (b). Panel (c) Densitometric analysis of TyrP patterns. The intensities of the bands of the control after 22 h of incubation were arbitrarily considered as 100%. Results are expressed as mean SEM, (n = 5).
(Langlais et al., 1988) may protect against oxidation for short periods of incubation; however, as proposed by Calamera et al. (2001), this protection is overwhelmed after 22 h in culture (Twigg et al., 1998b). In the presence of ROS, polyunsaturated fatty acids from the sperm plasma membrane are converted to lipid peroxide, modifying membrane fluidity and altering cell surface components by a chain reaction of LP function (Martınez & Morros, 1996). This study showed that in parallel with ReA, there is also a significant increase in LP (P < 0.05), with no detrimental effect on sperm viability (>80% viable cells). Sperm DNA fragmentation has been associated with ROS generation. The presence of H2O2 (25–200 lM) increases sperm DNA fragmentation (Aitken et al., 1998; Barroso et al., 2000), while the presence of substances with antioxidant properties in the medium protects DNA (Twigg et al., 1998b). In the present study, a baseline level of fragmented DNA of 15% was found after swimup, which is considered ‘naturally occurring DNA damage’ or ‘primary sperm damage’ commonly detected in semen (Gosalvez et al., 2011a). It is remarkable that even when spermatozoa were recovered directly from seminal plasma (with no centrifugation), a wide range of DNA damage was found (Twigg et al., 1998a). Three mechanisms associated with sperm damage in the ejaculate were described (Sakkas et al., 1999; Evenson & Wixon, 2008): alterations during histone/protamine replacement, apoptosis and oxidative stress (De Iuliis et al., 2009). The oxidative pathway was analysed and an oxidative challenge (H2O2) was introduced to guarantee DNA damage. The present study confirmed an increase in the 4
FI during sperm incubation, which reached a proportion of approximately 40% of fragmented cells and was not associated with any necrotic process (>80% viable cells at 22 h). This finding is in agreement with a study conducted by Gosalvez et al. (2011a), who reported a 1.6% per h increase in the FI, beginning after 6 h of incubation, with a higher rate of sperm DNA fragmentation in frozen samples. It has been proposed that those species lacking protamine 2 (P2) resisted fragmentation more efficiently during freezing/thawing or incubation conditions than those containing both protamines (P1 and P2) (L opez-Fernandez et al., 2007; Gosalvez et al., 2011b). Further studies are required to determine the molecular basis of this resistance or susceptibility to incubation. A significant increase in the FI (P < 0.05) was also observed after the induction of AR with hFF. The biochemical basis for this effect is probably mediated by the activation of different caspases and the progesterone (P)mediated elevation of intracytoplasmic Ca++ levels from intracellular stores via the nongenomic P receptor (Blackmore et al., 1990). This effect spills over into the mitochondria, inducing apoptosis-promoting factors released from the organelle into the cytoplasm, impairing sperm DNA (Lozano et al., 2009). Due to the fact that there is no ideal test with which to assess capacitation status, it has been widely accepted that cAMP, protein kinase A and tyrosine kinases appear to be involved in the intracellular signalling events associated with capacitation, which leads to the TyrP of a group of proteins (40–140 kDa) (Visconti et al., 2002). The present study showed that the TyrP of sperm proteins and the © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
J. Cicar e et al.
AR inducibility occur to a comparable extent in both H2O2-treated and nontreated spermatozoa, confirming that capacitation took place in the culture in parallel with DNA fragmentation. In conclusion, the results of the present study suggest that, in culture, spermatozoa undergo a restricted form of apoptosis but not necrosis, leading to the fragmentation of their DNA in the nucleus while conserving intracellular signalling events associated with the capacitation and AR required for fertilisation. Possibly, this situation may not exist in vivo because the female genital tract and its secretions provide a microenvironment that protects DNA integrity during the spermatozoa’s journey via many ‘prosurvivor factors’. However, special care should be taken in the case of patients with high rates of sperm DNA fragmentation at baseline, as the method used to prepare spermatozoa for ART can minimise or enhance the proportion of ROS generated. Prior evaluation of the in vitro increase in DNA fragmentation could be considered as a test to be included during the analysis of semen quality to determine how to manage those samples in which there is a tendency for the proportion of fragmented DNA to increase. References Agarwal A, Said TM (2003) Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 9:331–345. Aitken RJ (1995) Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev 7:659–668. Aitken RJ, Clarkson JS (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 81:459–469. Aitken RJ, Clarkson JS, Fishel S (1989) Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 40:183–197. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 59:1037–1046. Barroso G, Morshedi M, Oehninger S (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 15:1338–1344. Blackmore PF, Beebe SJ, Danforth DR, Alexander N (1990) Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 265:1376–1380. Calamera JC, Fernandez PJ, Buffone MG, Acosta AA, Doncel GF (2001) Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect. Andrologia 33:79–86.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Dynamic of DNA fragmentation
Calvo L, Vantman D, Banks SM, Tez on J, Koukoulis GN, Dennison L, Sherins RJ (1989) Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 52:1048–1054. Collins JA, Barnhart KT, Schlegel PN (2008) Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 89:823–831. De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, Nixon B, Aitken RJ (2009) DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy2’-deoxyguanosine, a marker of oxidative stress. Biol Reprod 81:517–524. Evenson DP, Wixon R (2008) Data analysis of two in vivo fertility studies using Sperm Chromatin Structure Assayderived DNA fragmentation index vs. pregnancy outcome. Fertil Steril 90:1229–1231. Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG (2003) The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Andrology 24:59–66. ~ez R, Fernandez JL, L Gosalvez J, N un opez-Fernandez C, Caballero P (2011a) Dynamics of sperm DNA damage in fresh versus frozen-thawed and gradient processed ejaculates in human donors. Andrologia 43:373–377. Gosalvez J, L opez-Fernandez C, Fernandez JL, Gouraud A, Holt WV (2011b) Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol Reprod Dev 78:951–961. de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C (1997) Reactive oxygen species and sperm physiology. Rev Reprod 2:48–54. Langlais J, Kan FW, Granger L, Raymond L, Bleau G, Roberts KD (1988) Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation. Gamete Res 20:185–201. Liu DY, Liu ML, Baker HW (2011) Quinn’s advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium. Reprod Biomed Online 3:735–739. L opez-Fernandez C, Crespo F, Arroyo F, Fernandez JL, Arana P, Johnston SD, Gosalvez J (2007) Dynamics of sperm DNA fragmentation in domestic animals II. The stallion. Theriogenology 68:1240–1250. Lozano GM, Bejarano I, Espino J, Gonzalez D, Ortiz A, Garcıa JF, Rodrıguez AB, Pariente JA (2009) Relationship between caspase activity and apoptotic markers in human sperm in response to hydrogen peroxide and progesterone. J Reprod Dev 55:615–621. Martınez P, Morros A (1996) Membrane lipid dynamics during human sperm capacitation. Front Biosc 1:d103–d117. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 4:31–37.
5
Dynamic of DNA fragmentation
Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ (1998a) Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod 4:439– 445. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ (1998b) Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA
6
J. Cicare et al.
fragmentation and effectiveness of antioxidants. Hum Reprod 13:1429–1436. Visconti PE, Westbrook VA, Chertihin O, Demarco I, Sleight S, Diekman AB (2002) Novel signaling pathways involved in sperm acquisition of fertilizing capacity. J Reprod Immunol 53:133–150. World Health Organization (2010) Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. WHO Press, Geneva.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation 1, A. Caille1, C. Zumoffen1, S. Ghersevich1, L. Bahamondes2 & M. J. Munuce1 J. Cicare 1 Laboratory of Reproductive Studies, Clinical Biochemistry Area, School of Biochemical and Pharmaceutical Sciences, National University of Rosario, Rosario, Argentina; 2 Human Reproduction Unit, Department of Obstetrics and Gynecology, School of Medical Sciences, University of Campinas (UNICAMP) and National Institute of Hormones and Women’s Health, Campinas, SP, Brazil
Keywords DNA fragmentation—dynamics—SCD test— sperm Correspondence Marıa Jos e Munuce, Laboratory of Reproductive Studies, Biochemical Chemistry Area, School of Biochemical and Pharmaceutical Sciences, University of Rosario, Suipacha 531, 2000 Rosario, Argentina. Tel.: + 54 341 4804597; Fax: + 54 341 4804592; E-mail: [email protected] Accepted: July 08, 2014 doi: 10.1111/and.12337
Summary The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham0 s F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.
Introduction Because of their aerobic metabolism, spermatozoa in culture generate low levels of reactive oxygen species (ROS), which include superoxide anion, hydrogen peroxide, hydroperoxyl radicals and hydroxyl radicals (Aitken, 1995). Many beneficial effects of ROS have been described, including their ability to stimulate hyperactivated motility and tyrosine phosphorylation in several proteins associated with capacitation (de Lamirande et al., 1997). However, high levels of ROS generated by infiltrating leucocytes or abnormal spermatozoa can overwhelm seminal plasma antioxidant defences, leading to the peroxidation of plasma membrane polyunsaturated fatty acids (LP) and DNA damage (Aitken & Clarkson, 1987). It is widely accepted that the presence of damaged DNA in mature ejaculated spermatozoa is associated with a range of adverse clinical outcomes including infertility, miscarriage and diseases in offspring (Agarwal & Said, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
2003; Collins et al., 2008). Moreover, a recent study reported on the ‘dynamics of sperm DNA fragmentation’ during in vitro incubation time, which was associated with a reduction in the fertilising potential (Gosalvez et al., 2011a). Considering that in most protocols of assisted reproductive technology (ART), selected human spermatozoa may remain incubated in vitro for several hours awaiting fertilisation (Liu et al., 2011). The aim of this study was to investigate the oxidative process associated with sperm incubation under capacitating conditions and its impact on DNA fragmentation and sperm function. Materials and methods The study was approved by the Institutional Review Board of the School of Biochemical and Pharmaceutical Sciences of the National University of Rosario, Argentina. Written informed consent that complies with all the 1
J. Cicare et al.
Dynamic of DNA fragmentation
Declaration of Helsinki Principles was signed by all the semen donors (n = 26). Semen sample and sperm processing Semen samples were obtained from healthy normozoospermic donors according to the World Health Organization guidelines (WHO, 2010). Only samples with 80% of viable cells. The ability to undergo the hFF-induced AR was also measured. Results showed that spontaneous and induced AR values were comparable in the control and H2O2-treated group, respectively: basal: 13.8 0.9% vs. induced AR: 40.6 4.6% and basal: 13.9 1.5% vs. induced AR: 37.8 5.4% (P < 0.001; n = 5). Furthermore, no differences were found in the TyrP pattern, either in the control or treatment groups, suggesting that capacitation had taken place during in vitro incubation in both situations (Fig. 4; NS; n = 5). Discussion
Fig. 1 Kinetics of redox activity (ReA) during sperm incubation under capacitating conditions. ReA was determined at 3, 6 and 22 h using the chemiluminescent probe luminol. Results are expressed as mean SEM, (n = 16). *(P < 0.05) when compared to semen and other incubation times.
Fig. 2 Kinetics of lipid peroxidation (LP) during sperm incubation under capacitating conditions. LP was measured using the thiobarbituric acid assay. Results are expressed as mean SEM, (n = 16). *(P < 0.05) when compared to semen and other incubation times.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
During sperm preparation for ART, gametes must be cocultured for several hours. Some forms of iatrogenic oxidative stress associated with the method used to prepare spermatozoa may induce DNA damage (Twigg et al., 1998a). The principal objective of the present study was to determine the kinetics of ReA and LP on spermatozoa in culture. No significant variation in ReA or LP levels was detected after 3 or 6 h of sperm incubation under optimal conditions. It is believed that the bovine serum albumin used as a cholesterol acceptor for capacitation
Fig. 3 Effect on DNA status during sperm incubation under capacitaing conditions and acrosome reaction. DNA was analysed using the sperm chromatin dispersion test. Results are expressed as mean SEM (n = 5). a,bP < 0.05 vs. control group, cP < 0.001 vs. post-swim-up, d,eP < 0.001 vs. post-swim-up and post-capacitation.
3
J. Cicare et al.
Dynamic of DNA fragmentation
(a)
(b)
(c)
Fig. 4 Analysis of phosphorylation on tyrosine residues. Panel (a) Proteins transferred to the blot (stained with Ponceau red) in the control (C) and treated groups (T). Panel (b) Immunodetection of TyrP using a monoclonal antiphosphotyrosine antibody. Molecular weight standards (MW) are indicated on the left side of Panel (b). Panel (c) Densitometric analysis of TyrP patterns. The intensities of the bands of the control after 22 h of incubation were arbitrarily considered as 100%. Results are expressed as mean SEM, (n = 5).
(Langlais et al., 1988) may protect against oxidation for short periods of incubation; however, as proposed by Calamera et al. (2001), this protection is overwhelmed after 22 h in culture (Twigg et al., 1998b). In the presence of ROS, polyunsaturated fatty acids from the sperm plasma membrane are converted to lipid peroxide, modifying membrane fluidity and altering cell surface components by a chain reaction of LP function (Martınez & Morros, 1996). This study showed that in parallel with ReA, there is also a significant increase in LP (P < 0.05), with no detrimental effect on sperm viability (>80% viable cells). Sperm DNA fragmentation has been associated with ROS generation. The presence of H2O2 (25–200 lM) increases sperm DNA fragmentation (Aitken et al., 1998; Barroso et al., 2000), while the presence of substances with antioxidant properties in the medium protects DNA (Twigg et al., 1998b). In the present study, a baseline level of fragmented DNA of 15% was found after swimup, which is considered ‘naturally occurring DNA damage’ or ‘primary sperm damage’ commonly detected in semen (Gosalvez et al., 2011a). It is remarkable that even when spermatozoa were recovered directly from seminal plasma (with no centrifugation), a wide range of DNA damage was found (Twigg et al., 1998a). Three mechanisms associated with sperm damage in the ejaculate were described (Sakkas et al., 1999; Evenson & Wixon, 2008): alterations during histone/protamine replacement, apoptosis and oxidative stress (De Iuliis et al., 2009). The oxidative pathway was analysed and an oxidative challenge (H2O2) was introduced to guarantee DNA damage. The present study confirmed an increase in the 4
FI during sperm incubation, which reached a proportion of approximately 40% of fragmented cells and was not associated with any necrotic process (>80% viable cells at 22 h). This finding is in agreement with a study conducted by Gosalvez et al. (2011a), who reported a 1.6% per h increase in the FI, beginning after 6 h of incubation, with a higher rate of sperm DNA fragmentation in frozen samples. It has been proposed that those species lacking protamine 2 (P2) resisted fragmentation more efficiently during freezing/thawing or incubation conditions than those containing both protamines (P1 and P2) (L opez-Fernandez et al., 2007; Gosalvez et al., 2011b). Further studies are required to determine the molecular basis of this resistance or susceptibility to incubation. A significant increase in the FI (P < 0.05) was also observed after the induction of AR with hFF. The biochemical basis for this effect is probably mediated by the activation of different caspases and the progesterone (P)mediated elevation of intracytoplasmic Ca++ levels from intracellular stores via the nongenomic P receptor (Blackmore et al., 1990). This effect spills over into the mitochondria, inducing apoptosis-promoting factors released from the organelle into the cytoplasm, impairing sperm DNA (Lozano et al., 2009). Due to the fact that there is no ideal test with which to assess capacitation status, it has been widely accepted that cAMP, protein kinase A and tyrosine kinases appear to be involved in the intracellular signalling events associated with capacitation, which leads to the TyrP of a group of proteins (40–140 kDa) (Visconti et al., 2002). The present study showed that the TyrP of sperm proteins and the © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
J. Cicar e et al.
AR inducibility occur to a comparable extent in both H2O2-treated and nontreated spermatozoa, confirming that capacitation took place in the culture in parallel with DNA fragmentation. In conclusion, the results of the present study suggest that, in culture, spermatozoa undergo a restricted form of apoptosis but not necrosis, leading to the fragmentation of their DNA in the nucleus while conserving intracellular signalling events associated with the capacitation and AR required for fertilisation. Possibly, this situation may not exist in vivo because the female genital tract and its secretions provide a microenvironment that protects DNA integrity during the spermatozoa’s journey via many ‘prosurvivor factors’. However, special care should be taken in the case of patients with high rates of sperm DNA fragmentation at baseline, as the method used to prepare spermatozoa for ART can minimise or enhance the proportion of ROS generated. Prior evaluation of the in vitro increase in DNA fragmentation could be considered as a test to be included during the analysis of semen quality to determine how to manage those samples in which there is a tendency for the proportion of fragmented DNA to increase. References Agarwal A, Said TM (2003) Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 9:331–345. Aitken RJ (1995) Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev 7:659–668. Aitken RJ, Clarkson JS (1987) Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 81:459–469. Aitken RJ, Clarkson JS, Fishel S (1989) Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 40:183–197. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 59:1037–1046. Barroso G, Morshedi M, Oehninger S (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 15:1338–1344. Blackmore PF, Beebe SJ, Danforth DR, Alexander N (1990) Progesterone and 17 alpha-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 265:1376–1380. Calamera JC, Fernandez PJ, Buffone MG, Acosta AA, Doncel GF (2001) Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect. Andrologia 33:79–86.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
Dynamic of DNA fragmentation
Calvo L, Vantman D, Banks SM, Tez on J, Koukoulis GN, Dennison L, Sherins RJ (1989) Follicular fluid-induced acrosome reaction distinguishes a subgroup of men with unexplained infertility not identified by semen analysis. Fertil Steril 52:1048–1054. Collins JA, Barnhart KT, Schlegel PN (2008) Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 89:823–831. De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, Nixon B, Aitken RJ (2009) DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy2’-deoxyguanosine, a marker of oxidative stress. Biol Reprod 81:517–524. Evenson DP, Wixon R (2008) Data analysis of two in vivo fertility studies using Sperm Chromatin Structure Assayderived DNA fragmentation index vs. pregnancy outcome. Fertil Steril 90:1229–1231. Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG (2003) The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Andrology 24:59–66. ~ez R, Fernandez JL, L Gosalvez J, N un opez-Fernandez C, Caballero P (2011a) Dynamics of sperm DNA damage in fresh versus frozen-thawed and gradient processed ejaculates in human donors. Andrologia 43:373–377. Gosalvez J, L opez-Fernandez C, Fernandez JL, Gouraud A, Holt WV (2011b) Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol Reprod Dev 78:951–961. de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C (1997) Reactive oxygen species and sperm physiology. Rev Reprod 2:48–54. Langlais J, Kan FW, Granger L, Raymond L, Bleau G, Roberts KD (1988) Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation. Gamete Res 20:185–201. Liu DY, Liu ML, Baker HW (2011) Quinn’s advantage fertilization medium enhances zona pellucida-induced acrosome reaction compared with human tubal fluid medium. Reprod Biomed Online 3:735–739. L opez-Fernandez C, Crespo F, Arroyo F, Fernandez JL, Arana P, Johnston SD, Gosalvez J (2007) Dynamics of sperm DNA fragmentation in domestic animals II. The stallion. Theriogenology 68:1240–1250. Lozano GM, Bejarano I, Espino J, Gonzalez D, Ortiz A, Garcıa JF, Rodrıguez AB, Pariente JA (2009) Relationship between caspase activity and apoptotic markers in human sperm in response to hydrogen peroxide and progesterone. J Reprod Dev 55:615–621. Martınez P, Morros A (1996) Membrane lipid dynamics during human sperm capacitation. Front Biosc 1:d103–d117. Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi PG, Bianchi U (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 4:31–37.
5
Dynamic of DNA fragmentation
Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ (1998a) Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod 4:439– 445. Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ (1998b) Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA
6
J. Cicare et al.
fragmentation and effectiveness of antioxidants. Hum Reprod 13:1429–1436. Visconti PE, Westbrook VA, Chertihin O, Demarco I, Sleight S, Diekman AB (2002) Novel signaling pathways involved in sperm acquisition of fertilizing capacity. J Reprod Immunol 53:133–150. World Health Organization (2010) Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. WHO Press, Geneva.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–6
