Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence

Alwin A.H.A. Derijck 1 2 Godfried W. van der Heijden 0 2 Liliana Ramos 2 Maud Giele 2 3 Jan A.M. Kremer 2 Peter de Boer 2 0 Present address: Carnegie Institution of Washington, Department of Embryology , 3520 San Martin Drive, Baltimore, MD 21218 , USA 1 Present address: University Medical Center Utrecht, Department of Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience , Universiteitsweg 100, 3584 CG, Utrecht , The Netherlands 2 Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre , PO Box 9101, 6500 HB Nijmegen , The Netherlands 3 Present address: Orthopedic Research Laboratory, Department of Orthopedics, Radboud University Nijmegen Medical Centre , PO Box 9101, 6500 HB Nijmegen , The Netherlands BACKGROUND: Among ICSI children de novo structural chromosome aberrations of male descent are increased. Misrepair of double-strand DNA breaks (DSBs) is a prerequisite for such aberrations to occur. To date, no absolute assessment of the number of DSBs in human sperm nuclei after gamete fusion has been described. METHODS: Using man-mouse heterologous ICSI and gH2AX immunofluorescent staining, capable of detecting a single DSB, the number of lesions in ICSI selected sperm from normozoospermic men (n 5 2) and oligozoospermic patients (n 5 3) was quantified. A comparison with a subfertile male mouse model (n 5 5) has been made. In addition, the fate of morphologically normal ejaculated immotile sperm after ICSI was examined. RESULTS: A significant increase in the fraction of sperm cells bearing DSBs was found in oligozoospermic semen compared with that from normozoospermic men (P < 0.01). The majority of morphologically normal immotile human sperm showed excess gH2AX staining and nuclear disintegration. However, some had a non-deviant DSB pattern. CONCLUSIONS: The increased fraction of DSB-positive sperm in both human and mouse oligozoospermic semen is adding to the surmise that semen from oligozoospermic patients has a reduced chromatin quality, causally related to reduced preimplantation embryo development. The use of ejaculated immotile sperm for in vitro reproduction is debatable due to sperm DNA degradation. - With the introduction of assisted reproduction techniques (ART), infertility no longer poses an absolute hurdle for couples to parent a child. In biological terms, ICSI is a less physiological form of ART, as this technique circumvents biological selection. As sperm selection is a poorly defined concept (Cohen and McNaughton, 1974) and ICSI is the sole option for many cases of male factor infertility, this poses a degree of concern. Sperm from such men usually meet several or all of the World Health Organization (WHO) criteria for oligo-astheno-teratozoospermia (OAT). One major concern pertains to an increased risk of de novo mutation at both the level of the gene and chromosome. An increased risk of de novo mutation at the chromosomal level has been indicated. *Both authors contributed equally. In sperm of OAT males, the normally low incidence of numerical chromosomal abnormalities is about doubled implying meiotic defects (Marchetti and Wyrobek, 2005), possibly illustrated by lower recombination rates (Gonsalves et al., 2004), giving rise to an increased number of aneuploid offspring (Bonduelle et al., 2002). The aetiology of structural chromosome aberrations, that are also more frequent among ICSI descendants (Bonduelle et al., 2002), remains unclear. For a long time, this class of mutation has been known to be largely of male descent (Olson and Magenis, 1988). Misrepair of double-strand DNA breaks (DSBs) is a prerequisite for structural chromosome aberrations to occur (Richardson and Jasin, 2000). Indeed, radiationinduced DSBs in mouse and human sperm lead to chromosome abnormalities, detectable in the paternal chromosomes at the first cleavage division (Matsuda et al., 1985; Kamiguchi and Tateno, 2002). DSBs are a characteristic of living cells in that they do occur spontaneously in the cell cycle during DNA replication and are instrumental in the generation of antibody diversity (Hoeijmakers, 2001) and meiotic recombination. If left unrepaired, DSBs would inhibit the cell cycle or alternatively would lead to gross structural alteration of the chromosome complement, most likely by a high frequency of deletions. Therefore a DSB is considered to be a most serious DNA lesion. Hence, special DNA repair pathways, notably homologous recombination (HR) repair and non-homologous end joining (NHEJ) evolved in the course of evolution to take care of such damage (see for review Wyman and Kanaar, 2006). Structural chromosome abnormalities such as dicentric chromosomes, reciprocal translocations and acentric fragments are conceptualized as failures of both NHEJ and HR, for which the word misrepair is coined. DSBs are also used to rearrange chromatin, a most prominent example being the elongation of round spermatids when the enzyme Topoisomerase II (Laberge and Boissonneault, 2005) both creates and religates these lesions. This breakage activity is implicated in the loss of the nucleosomal chromatin structure on the way to transition protein and protamine occupation of DNA in sperm. Sperm present a special group of nuclei with respect to DNA damage as nuclei are very compact. Yet, several methods have yielded indications for the presence of DNA damage at a larger scale than in somatic cell systems that rely on active DNA repair, that is not available to the sperm nucleus (Kofman-Alfaro and Chandley, 1971; Sega et al., 1978). For the past decades, the DNA integrity of the sperm nucleus has been measured by numerous techniques, i.e. in situ nick translation, terminal deoxynucleotidyl transferase dUTP end labelling (TUNEL), single-cell electrophoresis (SCE, or comet assay in alkaline and neutral variants), sperm chromatin dispersion test and sperm chromatin structure assay (SCSA) (Fernandez et al., 2005; Evenson and Wixon, 2006). Apart form the neutral comet assay (Van Kooij et al., 2004), these methods do not specifically sense DSBs. Moreover, the neutral comet assay does not generate absolute numbers of breaks. For the cytogenetic analysis of human sperm after heterologous insemination, hamster (Rudak et al., 1978) and mouse (Lee et al., 1996) secondary oocytes have been used in the past. Heterologous ICSI of mouse oocytes is a proven method for the assessment of oocyte activating power and chromosomal constitution of human sperm, mimicking the ICSI-involved sperm selection procedure (Rybouchkin et al., 1995; Lee et al., 1996). Frequencies of 1.3 and 6.9% for, respectively, numerical and structural chromosome aberrations have been found in morphologically normal semen using these methods (Lee et al., 1996). However, from the data on structural chromosome abnormalities, the absolute number of breaks per sperm nucleus cannot be deduced, due to an absence of knowledge regarding the reliability of repair mechanisms, i.e. the (...truncated)