Article Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis

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1 RBMOnline - Vol 7. No Reproductive BioMedicine Online; on web 16 July 2003 Article Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis Dr Denny Sakkas obtained his PhD at Monash University, Australia. Since then he has headed research groups and directed In Vitro Fertilization Laboratories at the University of Geneva, and Birmingham, UK, and currently in the Department of Obstetrics at Yale University. His research interests include: (i) understanding the role of apoptosis and presence of nuclear DNA damage in the spermatozoa of men with poor semen parameters; (ii) the consequences of using abnormal spermatozoa to fertilize eggs on the developing embryo and ensuing fetus; (iii) the role of glucose during gamete fusion; and (iv) examining for markers of preimplantation embryos, which help to predict subsequent viability. Dr Denny Sakkas Denny Sakkas 1,4, Emre Seli 1, Davide Bizzaro 2, Nicoletta Tarozzi 3, Gian Carlo Manicardi 3 1 Department of Obstetrics and Gynaecology, Yale University School of Medicine, CN, USA 2 Institute of Biology and Genetics, University of Ancona, Italy 3 Department of Animal Biology, University of Modena and Reggio Emilia, Italy 4 Correspondence: Yale University School of Medicine, Department of Obstetrics and Gynecology, 333 Cedar Street, New Haven, CT , USA. Fax: ; Denny.Sakkas@yale.edu Abstract The mechanisms responsible for producing abnormal spermatozoa in the ejaculate are relatively unknown. Numerous studies have now shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa and the abnormal persistence of apoptotic marker proteins. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the replacement of histones with protamines during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve knowledge about certain causes of male infertility. Keywords: abnormal spermatozoa, apoptosis, nuclear remodelling, spermatogenesis 428 Introduction Abnormal semen parameters remain one of the most common causes of subfertility amongst couples. In the human, a strong association has been found between abnormal semen parameters and the presence of nuclear DNA strand breaks in ejaculated spermatozoa. The presence of spermatozoa in the human ejaculate that have damaged nuclear DNA or loosely packaged chromatin has been confirmed by numerous studies (Evenson et al., 1980; Foresta et al., 1992; Bianchi et al., 1993; Gorczyca et al., 1993; Manicardi et al., 1995; Sailer et al., 1995; Sun et al., 1997; Lopes et al., 1998). A study by Host and co-workers (1999) also reported that DNA strand breaks were found significantly more often in spermatozoa from men with unexplained infertility compared with those from normal fertile men. One major area of understanding that needs to improve is if and how the testes function to detect and remove compromised spermatozoa from the process of spermatogenesis and the ejaculate. The process most likely delegated this task is apoptosis, or programmed cell death. Apoptosis and spermatogenesis Spermatogenesis is a complex process of proliferation and differentiation transforming spermatogonia into mature spermatozoa. This unique process involves a series of mitoses and meioses and results in the production of up to spermatozoa daily. It is estimated that cell death occurring during normal spermatogenesis in mammals results in the loss of up to 75% of the potential number of spermatozoa (Huckins, 1978). Investigations using animal models have shown that the overproliferation of early germ cells is tempered by selective apoptosis of their progeny (Allan et al., 1992; Tapanainen et al., 1993; Bartke, 1995; Billig et al., 1995; Furuchi et al.,

2 1996; Rodriguez et al., 1997; Sinha Hikim et al., 1997). Testicular germ cell apoptosis occurs normally and continuously throughout life. The aetiological factors and molecular mechanisms leading to apoptosis in testicular germ cells have not yet been completely understood. Many aetiological factors, including gonadotrophin withdrawal (Hikim et al., 1995), cryptorchidism (Shikone et al., 1994; Henriksen et al., 1996), heat exposure (Yin et al., 1997; Lue et al., 1999) and irradiation (Henriksen and Parvinen, 1998), have been shown to induce apoptosis in testicular germ cells. In the human, the function of apoptosis and its impact on the ejaculated spermatozoa is still not clear. Apoptosis can be postulated to have two putative roles during normal spermatogenesis: limitation of the germ cell population to numbers that can be supported by the Sertoli cells (Lee et al., 1997), and possibly selective depletion of abnormal spermatozoa. Although evidence exists indicating that the germ cell population is tempered by apoptosis, the role of apoptosis in the selective depletion of abnormal spermatozoa is not known. In a previous study, it has been found that men with abnormal sperm parameters display higher levels of the apoptotic protein Fas on their ejaculated spermatozoa (Sakkas et al., 1999b). The presence of Fas on ejaculated spermatozoa correlates strongly with a decreased sperm concentration and spermatozoa with abnormal morphology. More recently, several studies have also found that other apoptotic markers such as Bcl-x, p53 and annexin V are also present on ejaculated human spermatozoa and show distinct relationships with abnormal semen parameters (Barroso et al., 2000; Sakkas et al., 2002). In mouse models, numerous pro- and anti-apoptotic proteins have been found to play key roles in spermatogenesis. For example, over-expression of anti-apoptotic Bcl-2 or Bcl-x blocks cell death at a critical stage and results in disruption of normal spermatogenesis and infertility in mice (Furuchi et al., 1996; Rodriguez et al., 1997), while proapoptotic Bax is required for normal developmental germ cell death in type A mouse spermatogonia, specifically dividing (A2, A3, and A4) spermatogonia, at a time at which the number of spermatogonia is regulated in a density-dependent manner (Russell et al., 2002). The massive hyperplasia that occurs in Bax-deficient mice subsequently results in Bax-independent cell death, which may be triggered by overcrowding of the seminiferous epithelium. The relationship of the apoptotic proteins linked with poor semen parameters may indicate that indeed the spermatozoa that have DNA damage are also those that are positive for certain apoptotic proteins. When it was investigated whether terminal deoxynucleotidyl transferase-mediated dudp nickend labelling (TUNEL) expression was evident in the same samples that had high Fas, p53 or Bcl-x expression, it was found that there was no strict relationship. When double labelling was performed, to see if the same population of spermatozoa that were TUNEL-positive would also show either Bcl-x or p53 expression, it was found that although the majority of p53 positive spermatozoa also showed TUNEL positivity, only about half of the Bcl-x positive spermatozoa showed TUNEL positivity (Sakkas et al., 2002). It appears from these results that ejaculated spermatozoa exhibiting DNA damage do not always show distinct apoptotic markers. Escape of sperm cells earmarked for apoptosis It has previously been postulated that the presence of Fas on ejaculated human spermatozoa may be indicative of sperm cells that are earmarked for apoptosis, but they escape apoptosis because there are too many for the available Fas-L to induce apoptosis, or signalling through Fas is not functional. This has previously been called abortive apoptosis (Sakkas et al., 1999b). How these Fas-labelled spermatozoa escape apoptosis is unknown; however, the persistence of increased expression levels of some apoptotic markers on mature spermatozoa may involve a similar mechanism to that reported in the rat (Blanco-Rodriguez and Martinez-Garcia, 1999). Blanco-Rodriguez and Martinez-Garcia (1999) have shown that apoptotic signalling molecules can be restricted to a specific cytoplasmic compartment during rat spermatogenesis, and form residual bodies similar to apoptotic bodies. These residual bodies have increased expression levels of caspase-1, c-jun, p53 and p21. Although human spermatozoa do not normally possess residual bodies, they do sometimes retain cytoplasm. Therefore a defect in the cytoplasmic remodelling may be responsible for some of the mature spermatozoa showing high levels of apoptotic markers. Indeed, it has been found that a number of spermatozoa showing cytoplasmic retention also stain positively for the Bcl-x antibody, indicating that a similar phenomenon may be evident in human as reported in rat spermatozoa (Huszar and Sakkas, unpublished results). Cytoplasmic retention has already been shown to vary in human spermatozoa that exhibit different biochemical markers such as creatine kinase (Huszar et al., 1998, 2000; Gergely et al., 1999) and this has also been linked to increased aneuploidy frequencies in human spermatozoa (Kovanci et al., 2001). Faulty DNA remodelling during spermiogenesis In addition to the presence of apoptotic marker proteins such as Fas, the other key indicator believed to be indicative of apoptosis is the presence of nuclear DNA strand breaks in ejaculated spermatozoa. This may be due to factors unrelated to apoptosis. McPherson and Longo (1993b) proposed that the presence of endogenous nicks in ejaculated spermatozoa might be indicative of incomplete maturation during spermiogenesis. This was thought to come about when considering the remodelling of chromatin that takes place during spermiogenesis, and in particular the mechanisms that allow the replacement of histones by protamines during the spermatid stages. They postulated (McPherson and Longo, 1992, 1993a,b) that chromatin packaging might necessitate endogenous nuclease activity to create and ligate nicks that facilitate the replacement of the histones by protamine. The endogenous nuclease, topoisomerase II (topo II), may play a role in both creating and ligating nicks during spermiogenesis. Topo II enzymes function by transiently introducing DNA double strand breaks, allowing passage of one double helix through another, and resealing the double strand break (Wang et al., 1990). The presence of topoisomerase II in human testes indicates that the abnormal function of this nuclease may exacerbate the presence of DNA strand breaks in the ejaculated spermatozoon (unpublished results). 429

3 Abnormal spermatozoa in the ejaculate The two mechanisms described above, either individually or together, have some bearing on the presence of abnormal spermatozoa in the ejaculate. These two mechanisms may or may not be interrelated. The following model is proposed to explain the presence of nuclear DNA damage and apparent anomalies in apoptosis during human spermatogenesis (see Figure 1). As in other mammalian systems, it appears that the Fas-mediated system and its related pathways are responsible during the germ cell stages for controlling spermatogenesis (Pentikainen et al., 1999). In addition, the Bcl-2 family of proteins, in particular that were activated in the earlier stages. The extensive cytoplasmic remodelling that occurs during the later stages of spermatogenesis may also disrupt the apoptotic pathways that are functional prior to spermiogenesis. A different population of sperm could exist that have escaped programmed cell death and express various apoptotic markers, a procedure termed abortive apoptosis (Sakkas et al., 1999b). Having this model in mind, the apparition of defective spermatozoa in the ejaculate could be related to anomalies in the repair of the DNA breaks that appear in spermatids and/or increased apoptosis during early spermatogenesis. The two processes may be operating independently but they also may obstruct the function of each other. Figure 1. A model of the relationship between apoptosis and cellular remodelling during spermatogenesis arising in normal mature spermatozoa and abnormal spermatozoa. Abnormal spermatozoa are thought to arise due to failed apoptosis and/or a failure to repair DNA strand breaks appearing during the early spermatid stage. Abnormal spermatogonia are earmarked for apoptosis and undergo normal apoptosis. Otherwise, a normal spermatozoon is produced from spermatogonia that progress during spermatogenesis and undergo histone protamine exchange during spermiogenesis. During abnormal spermatogenesis, a spermatogonia is earmarked for apoptosis but escapes the process, leading to possible combinations of anomalies including histone protamine exchange, DNA remodelling and also cytoplasmic and membrane abnormalities. 430 Bcl-x, would also be a candidate in the selection process of which cells proceed to maturity (Sakkas et al., unpublished results). These systems would have their greatest influence pre-spermiogenesis, prior to the remodelling of the nucleus that takes place during spermiogenesis and the exclusion of the cytoplasm. During spermiogenesis, DNA breaks appear normally in the early spermatids and it is predicted that faulty nuclear remodelling also affects the nature of the ejaculated spermatozoa (Sakkas et al., 1999a). Furthermore, the nuclear remodelling that takes place during spermiogenesis could also change or derail the classic programmed cell death pathway/s Conclusion Anomalies in one or both of these pathways would lead to the presence of abnormal spermatozoa in the ejaculate. Failure of apoptosis to selectively deplete those sperm cells earmarked for programmed cell death would lead to them escaping the process, even if they had already partially commenced the process. The final outcome would be the production of spermatozoa that possess a range of anomalies. This would include spermatozoa that have single or combinations of abnormalities including abnormal levels of apoptotic proteins

4 and/or cytoplasmic retention, abnormal chromatin packaging (indicated by low levels of protamine) and the presence of DNA strand breaks. Finally, it may be the same abnormal spermatozoa, produced after spermatogenesis, that are more susceptible to environmental factors and oxidative attack (Aitken et al., 2003; Aitken and Krausz, 2001). References Aitken RJ, Krausz C 2001 Oxidative stress, DNA damage and the Y chromosome. Reproduction 122, Aitken RJ, Baker MA, Sawyer D 2003 Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease. Reproductive BioMedicine Online 7, Allan D, Harmon B, Roberts S 1992 Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat. Cellular Proliferation 25, Barroso G, Morshedi M, Oehninger S 2000 Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. 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