An estimated 15% of all couples are affected by a clinical degree of infertility. In approximately 50% of these couples successful conception is affected by the male factor. Among factors that can negatively affect male fertility are genetic disorders, infectious diseases, physical obstruction of reproductive organs, or exposure of men to environmental or medical toxicants as during a cancer therapy regimen. If spermatogenesis is performed, the degree of chromatin integrity and frequency of residual strand breaks in mature sperm cells have been identified as important parameters in infertility patients and, of course, healthy men as well. All in all, chromatin integrity has been identified as an important prerequisite for normal sperm cell function. Incomplete chromatin condensation and the presence of persistent DNA strand breaks indicate a severe reduction of the fertilization potential in mature spermatozoa. Lack of nuclear integrity often results from faulty or incomplete execution of chromatin remodeling steps and DNA strand break management in immature steps of spermatid development. In spite of the widely appreciated importance of nuclear integrity for successful transmission of the male genetic information, the mechanisms that are involved in achieving and maintaining this adequate nuclear composition are still not fully understood. In somatic cells, poly(ADP-ribose) metabolism is one of the key pathways in the maintenance of genetic integrity and we have recently discovered novel evidence suggesting crucial functions of the pathway in male germ cell development.
In brief, we use knock-out mouse models to investigate essential chromatin remodeling steps in germ cell development, mitotic and meiotic spindle organization, chromosome segregation but also to the regulation of apoptosis when things go awry. The involvement of poly(ADP-ribose) metabolism, which is mediated by up to 18 known polymerases (PARPs) and a single catabolic enzyme, PARG (see below for explanation), is a major focus of our research, including the elucidation of emerging signalling functions of the polymer and its specific binding proteins.
A little bit of general background information on the Poly(ADP-ribos)ylation pathway:
DNA strand breaks directly and immediately activate the ubiquitous nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1, Figure 1). Upon activation, PARP-1 forms a large, branched ADP-ribose polymer by cleaving NAD+ into nicotinamide and ADP-ribose (ADPR), which becomes polymerized and covalently attached to proteins such as PARP-1 itself as an automodification reaction. Examples of other target proteins for this posttranslational modification are histones, p53 and DNA ligase III. Breakdown of the unique biopolymer is facilitated by poly(ADP-ribose) glycohydrolase (PARG) with ADPR polymer turnover being a dynamic process mediated by the interplay of PARP-1 and PARG. Automodified PARP-1 is catalytically inactive and is released from the DNA strand break. The ability of PARP-1 to bind to DNA strand breaks and to become activated again is restored upon removal of the ADPR polymer by PARG. The resulting shuttling mechanism mediated by PARP-1 and PARG is also the basis for the transient removal of histones and other DNA binding proteins from the DNA, which results in a short-term local chromatin decondensation (Figure 2). PARP-1 has important protective roles in DNA repair, chromatin modulation and cellular recovery after genotoxic insults and has therefore also been named a “guardian of the genome”. Inhibition of PARP-1 has been shown to strongly decrease genomic stability and to cause hypersensitivity to genotoxic agents. Our preliminary data indicate for the first time that PARP-1 is a highly active enzyme during normal chromatin reorganization in elongating spermatids coincident with DNA breaks that occur during the chromatin remodeling steps required for successful spermiogenesis (Figure 3). The occurrence of controlled DNA strand breaks in this process has been described to facilitate the transition from the supercoiled histone form to the linear protamine form in spermatids.
Figure 1: DNA strand break dependent poly(ADP-ribose) metabolism as mediated by PARP-1 and PARG. Please note that i) there are altogether up to 18 PARP enzymes with vastly different functions, modes of activation and specific activities and ii) NAD+ is not used here as a classic coenzyme for a redox reaction (NAD+/NADH) but is actually used as a substrate in a depleting fashion.
Figure
2: A simplified
model of poly(ADP-ribos)ylation dependent histone shuttling
providing a hypothetical view of local chromatin
reorganization afforded by PARP-1, PARP-2 and PARG.
Figure 3: Step-specific poly(ADP-ribose) formation in rat spermatid development.
Selected Publications
Meyer R, Nagl W (1993). Endopolyploidy in the extraembryonic membranes of the snake, Elaphe guttata. Protoplasma 172: 132-135.
Beneke S, Meyer R, Burkle A (1997). Isolation of cDNA Encoding Full-Length Rat (Rattus norvegicus) Poly(ADP-Ribose) Polymerase. Biochem Mol Biol Int 43:(4) 775-761
Meyer R, Muller M, Beneke S, Kupper JH, Burkle A (2000). Negative regulation of alkylation-induced sister-chromatid exchange by poly(ADP-ribose) polymerase-1 activity. Int J Cancer 88(3):351-5.
Meyer RG, Meyer-Ficca ML, Jacobson EL, Jacobson MK (2003). Human Poly(ADP-ribose) Glycohydrolase (PARG) Gene Structure and the Common Promoter Sequence it shares with Inner Mitochondrial Membrane Translocase 23 (TIM23). Gene 314: 181-190.
Meyer-Ficca ML, Meyer RG, Jacobson EL, Jacobson MK (2004). Human poly(ADP-ribose) glycohydrolase (PARG) is expressed in alternative splice variants which localize to different cell compartments. First two authors contributed equally. Exp Cell Res 297: 521-532.
Cortes U, Tong WM, Coyle DL, Meyer-Ficca ML, Meyer RG, Petrilli V, Herceg Z, Jacobson EL, Jacobson MK, Wang ZQ (2004). Disruption of the gene encoding poly(ADP-ribose) glycohydrolase (PARG) increases sensitivity to genotoxic and endotoxic stress. Mol Cell Bio. 24(16):7163-78.
Meyer-Ficca ML, Meyer RG, Kaiser H, Brack AR, Kandolf R, Küpper, J-H (2004). Comparative Analysis of inducible Expression Systems in transient Transfection Studies. Anal. Biochem. 334: 9–19
Meyer-Ficca ML, Scherthan H, Bürkle A, Meyer RG (2005). Poly(ADP-ribosyl)ation during chromatin remodeling steps in rat spermiogenesis. Chromosoma 114: 67–74
Meyer RG, Meyer-Ficca ML, Whatcott CJ, Jacobson EL, Jacobson MK. (2007). Two small enzyme isoforms mediate mammalian mitochondrial poly(ADP-ribose) glycohydrolase (PARG) activity. Exp. Cell Res. 313:2920-36.