Abstract
Sperm cell apoptosis is an energy and protein dependent process that results in sperm DNA damage. PARP cleavage (cPARP) is stimulated by DNA breaks for help in repair process, so it is considered as an early marker for apoptosis. Staurosporine (STS) is a PARP inducer while 3-aminobenzamide (3ABA), is a PARP inhibitor. Sperm DNA damage due to oxidative stress is considered as one of the causes of male infertility. Conflicting reports exist regarding the presence of whole or cPARP in human ejaculated spermatozoa. We evaluated: 1) the presence of cPARP in mature ejaculated spermatozoa and 2) if we can control its level in sperm fraction using apoptosis inducers with or without PARP inhibitor. Prospective-controlled study. Semen samples were collected from 10 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation. Mature and immature sperm fractions were resuspended into HTF media and divided in 5 aliquots (control, H2O2 treated, H2O2 + 3ABA, STS treated, and STS + 3ABA) incubated at 37°C with 5 % CO2 for 1 hour. The final concentrations were 2.5 μM for STS, 30 μM for H2O2, and 0.3 mM for 3ABA. After incubation, the mature and immature aliquots were evaluated for the early and late apoptosis by 1) annexin V, activated caspase-3, 2) sperm DNA damage by TUNEL and 3) cPARP by FACS. cPARP was detected in both neat and mature fractions. There were no significant differences between mature versus immature fractions regarding most of molecular apoptosis markers for treated and untreated sperm fractions for cPARP modulation.Table 1Correlation between the measured parameters in different treatment groupsUntreated sperm fractionsTreatmentActivated Caspase-3 (a C-3)TUNEL + sperm%Cleaved PARP + %Early apoptotic sperm %Late apoptotic sperm %rPrPrPrPrPSTS treated sperm0.960.120.30.50.720.90.420.130.60.12STS + 3ABA treated sperm0.940.0040.60.570.80.170.240.040.16<0.001H2O2 treated sperm0.940.110.80.20.80.26-0.190.002-0.13<0.001H2O2 + 3ABA treated sperm0.970.0020.510.560.81-0.7-0.20.003-0.06<0.001Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Open table in a new tab Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Cleaved PARP is present in neat as well as in washed ejaculated human spermatozoa. Its level is related to the extent of sperm DNA damage. PARP inhibitors can not prevent/repair oxidative or chemical-induced sperm DNA damage.
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