Reproductive Ageing: Current insights and a potential role of NAD in the reproductive health of aging fathers and their children.

In brief: In light of the increasing age of first-time fathers, this article summarizes the current scientific knowledge base on reproductive aging in the male, including sperm quality and health impacts for the offspring. The emerging role of NAD decline in reproductive aging is highlighted. Abstract: Over the past decades, the age of first-time fathers has been steadily increasing due to socio-economic pressures. While general mechanisms of aging are subject to intensive research, male reproductive aging has remained an understudied area, and the effects of increased age on the male reproductive system are still only poorly understood, despite new insights into the potential dire consequences of advanced paternal age for the health of their progeny. There is also growing evidence that reproductive aging is linked to overall health in men, but this review mainly focuses on pathophysiological consequences of old age in men, such as low sperm count and diminished sperm genetic integrity, with an emphasis on mechanisms underlying reproductive aging. The steady decline of NAD levels observed in aging men represents one of the emerging concepts in that regard. Because it offers some mechanistic rationale explaining the effects of old age on the male reproductive system, some of the NAD-dependent functions in male reproduction are briefly outlined in this review. The overview also provides many questions that remain open about the basic science of male reproductive aging.

Beyond Pellagra-Research Models and Strategies Addressing the Enduring Clinical Relevance of NAD Deficiency in Aging and Disease.

Research into the functions of nicotinamide adenine dinucleotide (NAD) has intensified in recent years due to the insight that abnormally low levels of NAD are involved in many human pathologies including metabolic disorders, neurodegeneration, reproductive dysfunction, cancer, and aging. Consequently, the development and validation of novel NAD-boosting strategies has been of central interest, along with the development of models that accurately represent the complexity of human NAD dynamics and deficiency levels. In this review, we discuss pioneering research and show how modern researchers have long since moved past believing that pellagra is the overt and most dramatic clinical presentation of NAD deficiency. The current research is centered on common human health conditions associated with moderate, but clinically relevant, NAD deficiency. In vitro and in vivo research models that have been developed specifically to study NAD deficiency are reviewed here, along with emerging strategies to increase the intracellular NAD concentrations.

Endogenous metabolism in endothelial and immune cells generates most of the tissue vitamin B3 (nicotinamide).

In mammals, nicotinamide (NAM) is the primary NAD precursor available in circulation, a signaling molecule, and a precursor for methyl-nicotinamide (M-NAM) synthesis. However, our knowledge about how the body regulates tissue NAM levels is still limited. Here we demonstrate that dietary vitamin B3 partially regulates plasma NAM and NAM-derived metabolites, but not their tissue levels. We found that NAD de novo synthesis from tryptophan contributes to plasma and tissue NAM, likely by providing substrates for NAD-degrading enzymes. We also demonstrate that tissue NAM is mainly generated by endogenous metabolism and that the NADase CD38 is the main enzyme that produces tissue NAM. Tissue-specific CD38-floxed mice revealed that CD38 activity on endothelial and immune cells is the major contributor to tissue steady-state levels of NAM in tissues like spleen and heart. Our findings uncover the presence of different pools of NAM in the body and a central role for CD38 in regulating tissue NAM levels.

Low NAD+ Levels Are Associated With a Decline of Spermatogenesis in Transgenic ANDY and Aging Mice.

Advanced paternal age has increasingly been recognized as a risk factor for male fertility and progeny health. While underlying causes are not well understood, aging is associated with a continuous decline of blood and tissue NAD+ levels, as well as a decline of testicular functions. The important basic question to what extent ageing-related NAD+ decline is functionally linked to decreased male fertility has been difficult to address due to the pleiotropic effects of aging, and the lack of a suitable animal model in which NAD+ levels can be lowered experimentally in chronologically young adult males. We therefore developed a transgenic mouse model of acquired niacin dependency (ANDY), in which NAD+ levels can be experimentally lowered using a niacin-deficient, chemically defined diet. Using ANDY mice, this report demonstrates for the first time that decreasing body-wide NAD+ levels in young adult mice, including in the testes, to levels that match or exceed the natural NAD+ decline observed in old mice, results in the disruption of spermatogenesis with small testis sizes and reduced sperm counts. ANDY mice are dependent on dietary vitamin B3 (niacin) for NAD+ synthesis, similar to humans. NAD+-deficiency the animals develop on a niacin-free diet is reversed by niacin supplementation. Providing niacin to NAD+-depleted ANDY mice fully rescued spermatogenesis and restored normal testis weight in the animals. The results suggest that NAD+ is important for proper spermatogenesis and that its declining levels during aging are functionally linked to declining spermatogenesis and male fertility. Functions of NAD+ in retinoic acid synthesis, which is an essential testicular signaling pathway regulating spermatogonial proliferation and differentiation, may offer a plausible mechanism for the hypospermatogenesis observed in NAD+-deficient mice.

Targeting PARP11 to avert immunosuppression and improve CAR T therapy in solid tumors.

Evasion of antitumor immunity and resistance to therapies in solid tumors are aided by an immunosuppressive tumor microenvironment (TME). We found that TME factors, such as regulatory T cells and adenosine, downregulated type I interferon receptor IFNAR1 on CD8+ cytotoxic T lymphocytes (CTLs). These events relied upon poly-ADP ribose polymerase-11 (PARP11), which was induced in intratumoral CTLs and acted as a key regulator of the immunosuppressive TME. Ablation of PARP11 prevented loss of IFNAR1, increased CTL tumoricidal activity and inhibited tumor growth in an IFNAR1-dependent manner. Accordingly, genetic or pharmacologic inactivation of PARP11 augmented the therapeutic benefits of chimeric antigen receptor T cells. Chimeric antigen receptor CTLs engineered to inactivate PARP11 demonstrated a superior efficacy against solid tumors. These findings highlight the role of PARP11 in the immunosuppressive TME and provide a proof of principle for targeting this pathway to optimize immune therapies.

Metabolism in Male Reproductive Aging.

Similar to female reproductive health, male reproductive health declines with increasing age, albeit in a more gradual way. In the US, the average age of first-time fathers has been steadily increasing since 1980. This is concerning because increasing paternal age is positively correlated with reduced sperm chromatin quality and higher numbers of DNA strand breaks (DNA sb), which negatively affects pregnancy outcome and child development. While underlying reasons are not well understood, one of the well-known hallmarks of aging is a significant decline of body nicotinamide adenine dinucleotide (NAD) levels. We propose that low body-wide NAD levels provide a plausible explanation for metabolic alterations that are associated with declining hormonal production and testicular volume, as well as reduced sperm quality in aging men.

In vitro effects of poly(ADP-ribose) polymerase inhibitors on the production of tumor necrosis factor-α by interferon- γ - and lipopolysaccharide-stimulated peripheral blood mononuclear cells of horses.

OBJECTIVE: To evaluate effects of poly(ADP-ribose) polymerase-1 (PARP1) inhibitors on the production of tumor necrosis factor-α (TNF-α) by interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of horses as an in vitro model of inflammation in horses. SAMPLE: 1,440 samples of PBMCs from 6 healthy research horses. PROCEDURES: From heparinized whole blood samples, PBMC cultures were obtained. An initial dose-response trial on 48 PBMC samples from 2 horses (24 samples each) was used to determine concentrations of IFN-γ and LPS for use as low- and high-level stimulation concentrations. Seventy-two PBMC samples from 6 horses were assigned equally to 1 of 4 PARP1 inhibition categories: no PARP1 inhibitor (PARP1 inhibition control); 2-((R)-2-methylpyrrolidin-2-yl)-1H-benzimidazole-4-carbozamide dihydrochloride (ABT888);4-(3-(1-(cyclopropanecarbonyl)piperazine-4-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one (AZD2281); or N-(6-oxo-5,6-dihydrophenanthridin-2-yl) -N,N-dimethylacetamide hydrochloride (PJ34). Samples of PBMCs from each horse and each PARP1 inhibition category were then assigned to 1 of 3 levels of IFN-γ and LPS stimulation: none (control), low stimulation, or high stimulation. After a 24-hour incubation period, a TNF-α ELISA was used to measure TNF-α concentration in the supernatant. Results were compared across treatments and for each horse. Data were analyzed with repeated-measures ANOVA. RESULTS: Median TNF-α concentration was significantly lower for PJ34-treated, high-level stimulated PBMCs than for PARP1 inhibition control, high-level stimulated PBMCs; however, no other meaningful differences in TNF-α concentration were detected among the inhibition and stimulation combinations. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that PJ34 PARP1 inhibition may reduce TNF-α production in horses, a potential benefit in reducing inflammation and endotoxin-induced damage in horses.

Alpha-Amino-Beta-Carboxy-Muconate-Semialdehyde Decarboxylase Controls Dietary Niacin Requirements for NAD+ Synthesis.

NAD+ is essential for redox reactions in energy metabolism and necessary for DNA repair and epigenetic modification. Humans require sufficient amounts of dietary niacin (nicotinic acid, nicotinamide, and nicotinamide riboside) for adequate NAD+ synthesis. In contrast, mice easily generate sufficient NAD+ solely from tryptophan through the kynurenine pathway. We show that transgenic mice with inducible expression of human alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase (ACMSD) become niacin dependent similar to humans when ACMSD expression is high. On niacin-free diets, these acquired niacin dependency (ANDY) mice developed reversible, mild-to-severe NAD+ deficiency, depending on the nutrient composition of the diet. NAD deficiency in mice contributed to behavioral and health changes that are reminiscent of human niacin deficiency. This study shows that ACMSD is a key regulator of mammalian dietary niacin requirements and NAD+ metabolism and that the ANDY mouse represents a versatile platform for investigating pathologies linked to low NAD+ levels in aging and neurodegenerative diseases.

Assessment of Poly(ADP-ribose) Polymerase1 (PARP1) expression and activity in cells purified from blood and milk of dairy cattle.

Poly(ADP-ribosyl)ation (PAR) is a post-translational protein modification catalysed by enzyme member of the poly(ADP-ribose) polymerases (PARPs) family. The activation of several PARPs is triggered by DNA strand breakage and the main PARP enzyme involved in this process is PARP1. Besides its involvement in DNA repair, PARP1 is involved in several cellular processes including transcription, epigenetics, chromatin re-modelling as well as in the maintenance of genomic stability. Moreover, several studies in human and animal models showed PARP1 activation in various inflammatory disorders. The aims of the study were (1) to characterize PARP1 expression in bovine peripheral blood mononuclear cells (PBMC) and (2) to evaluate PAR levels as a potential inflammatory marker in cells isolated from blood and milk samples following different types of infection, including mastitis. Our results show that (i) bovine PBMC express PARP1; (ii) lymphocytes exhibit higher expression of PARP1 than monocytes; (iii) PARP1 and PAR levels were higher in circulating PBMCs of infected cows; (iv) PAR levels were higher in cells isolated from milk with higher Somatic Cell Counts (SCC > 100,000 cells/mL) than in cells from milk with low SCCs. In conclusion, these findings suggest that PARP1 is activated during mastitis, which may prove to be a useful biomarker of mastitis.

Early histone H4 acetylation during chromatin remodeling in equine spermatogenesis.

Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residues K5, K8, and K12 in round spermatids almost immediately after meiotic division. Time courses of transition protein 1 (TP1), protamine 1, H2A histone family member Z (H2AFZ), and testis-specific histone H2B variant (TH2B) expression in stallion spermatogenesis were similar to the mouse where protamine 1 and TP1 were only expressed in elongating spermatids much later in spermatid development. The additional acetylation of H4 in K16 position (H4K16ac) was detected during a brief phase of spermatid elongation in both species, concomitant with the phosphorylation of the noncanonical histone variant H2AFX resulting from DNA strand break-mediated DNA relaxation. The results suggest that H4K16 acetylation, which is dependent on DNA damage signaling, may be more important for nucleosome replacement in spermiogenesis than indicated by data obtained in rodents and highlight the value of the stallion as an alternative animal model for investigating human spermatogenesis. A revised classification system of the equine spermatogenic cycle for simplified comparison with the mouse is proposed to this end.

Heritable sperm chromatin epigenetics: a break to remember.

Sperm chromatin not only has a unique structure to condense and protect the paternal DNA in transit, but also provides epigenetic information that supports embryonic development. Most of the unique sperm nuclear architecture is formed during the sweeping postmeiotic chromatin remodeling events in spermiogenesis, where the majority of nucleosomes are removed and replaced by protamines. The remaining histones and other chromatin proteins are located in structurally and transcriptionally relevant positions in the genome and carry diverse post-translational modifications relevant to the control of embryonic gene expression. How such postmeiotic chromatin-based programming of sperm epigenetic information proceeds, and how susceptible the process is to modulation by exogenous factors are key questions for understanding the inheritance of acquired epigenetic marks through the male germ line. We propose that transient DNA strand breaks mediated by topoisomerase II beta and the subsequent activation of DNA damage response pathways result in defined post-translational modifications of histones in spermiogenesis. These pathways, likely along with others, may contribute to chromatin remodeling in elongating spermatids, influence chromatin-based intergenerational inheritance of epigenetic information, and may be defective in pathologies of abnormal male gametogenesis and infertility.

The activity and inhibition of poly(ADP-ribose) polymerase-1 in equine peripheral blood mononuclear cells in vitro.

OBJECTIVES: To evaluate the poly (ADP-ribose) polymerase-1 (PARP1) enzyme and its inhibition in horses and explore its potential as a novel therapeutic target for equine intestinal ischemia-reperfusion injury by (1) identifying poly (ADP-ribose) (PAR) as an indication of PARP1 activation in equine cells using available immunoblot analytical techniques, (2) inducing PARP1 activation in an in vitro oxidative DNA damage model, (3) and demonstrating the inhibition of PARP1 in equine cells using commercially available PARP1 inhibitors. DESIGN: Experimental study. ANIMALS: Blood samples were collected from systemically healthy ponies (n = 3) and horses (n = 3). INTERVENTIONS: (1) Equine peripheral blood mononuclear cells were exposed to 3 different concentrations of hydrogen peroxide (H2 O2 ) and were lysed at specific time points. PARP1 activity was then assessed by using immunoblot analyses to determine PAR levels. (2) Equine peripheral blood mononuclear cells were preincubated with defined concentrations of PARP1 inhibitors prior to H2 O2 -mediated PARP1 stimulation. PAR levels reflecting PARP1 activity were determined using immunoblot analyses. MEASUREMENTS AND MAIN RESULTS: Commercially available anti-PAR antibodies were used successfully to identify equine PAR. There was a significant increase in PAR accumulation following treatment with H2 O2 . All of the tested PARP inhibitors significantly reduced PAR accumulation to or below basal levels following treatment with H2 O2 . CONCLUSIONS: This proof of principle study demonstrated that PAR, an indicator of PARP1 activity, can be identified in the equine species using immunoblot techniques, that equine PARP1 can be activated by H2 O2 -induced DNA damage, and that this activation can be inhibited by PARP1 enzyme inhibitors. The data suggest that the PARP1 pathway plays a role in the equine cellular response to oxidative DNA damage and supports its potential as a novel therapeutic target. Further research documenting an increase in PAR levels in vivo and the efficacy of PARP1 inhibitors in an equine intestinal ischemia-reperfusion model is needed.

Characterization of BRD4 during mammalian postmeiotic sperm development.

During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis.

Spermatid head elongation with normal nuclear shaping requires ADP-ribosyltransferase PARP11 (ARTD11) in mice.

Sperm are highly differentiated cells characterized by their species-specific nuclear shapes and extremely condensed chromatin. Abnormal head shapes represent a form of teratozoospermia that can impair fertilization capacity. This study shows that poly(ADP-ribose) polymerase-11 (ARTD11/PARP11), a member of the ADP-ribosyltransferase (ARTD) family, is expressed preferentially in spermatids undergoing nuclear condensation and differentiation. Deletion of the Parp11 gene results in teratozoospermia and male infertility in mice due to the formation of abnormally shaped fertilization-incompetent sperm, despite normal testis weights and sperm counts. At the subcellular level, PARP11-deficient elongating spermatids reveal structural defects in the nuclear envelope and chromatin detachment associated with abnormal nuclear shaping, suggesting functional relevance of PARP11 for nuclear envelope stability and nuclear reorganization during spermiogenesis. In vitro, PARP11 exhibits mono(ADP-ribosyl)ation activity with the ability to ADP-ribosylate itself. In transfected somatic cells, PARP11 colocalizes with nuclear pore components, such as NUP153. Amino acids Y77, Q86, and R95 in the N-terminal WWE domain, as well as presence of the catalytic domain, are essential for colocalization of PARP11 with the nuclear envelope, but catalytic activity of the protein is not required for colocalization with NUP153. This study demonstrates that PARP11 is a novel enzyme important for proper sperm head shaping and identifies it as a potential factor involved in idiopathic mammalian teratozoospermia.

Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression.

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo.

Separation of spermatogenic cell types using STA-PUT velocity sedimentation.

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types--both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa)--studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells--in this case, from the testes--through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 10(8) cells/spermatogenic cell type from a starting population of 7-8 x 10(8) cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

Alteration of poly(ADP-ribose) metabolism affects murine sperm nuclear architecture by impairing pericentric heterochromatin condensation.

The mammalian sperm nucleus is characterized by unique properties that are important for fertilization. Sperm DNA retains only small numbers of histones in distinct positions, and the majority of the genome is protamine associated, which allows for extreme condensation and protection of the genetic material. Furthermore, sperm nuclei display a highly ordered architecture that is characterized by a centrally located chromocenter comprising the pericentromeric chromosome regions and peripherally positioned telomeres. Establishment of this unique and well-conserved nuclear organization during spermiogenesis is not well understood. Utilizing fluorescence in situ hybridization (FISH), we show that a large fraction of the histone-associated sperm genome is repetitive in nature, while a smaller fraction is associated with unique DNA sequences. Coordinated activity of poly(ADP-ribose) (PAR) polymerase and topoisomerase II beta has been shown to facilitate DNA relaxation and histone to protamine transition during spermatid condensation, and altered PAR metabolism is associated with an increase in sperm histone content. Combining FISH with three-dimensional laser scanning microscopy technology, we further show that altered PAR metabolism by genetic or pharmacological intervention leads to a disturbance of the overall sperm nuclear architecture with a lower degree of organization and condensation of the chromocenters formed by chromosomal pericentromeric heterochromatin.

Genetic approaches to targeting multiple PARP genes in a mammalian genome.

The aim of this chapter is to present feasible strategies for producing novel Parp knock out mice as well multiple knock outs utilizing genetrap and specifically gene targeted ES cell clones publically available through international programs such as KOMP and IGTC. Specifically, we first describe general considerations and strategic decisions that precede the generation of knock out mice using these available materials, and an overview over clones relevant to the PARP family is provided. Detailed protocols for splice variant analysis of the gene of interest, to determine what to expect from a given gene trap clone, are presented. Furthermore, we provide a detailed and widely applicable step by step method to fine-map genomic genetrap insertion sites once targeted clones have been obtained. This is a prerequisite for development of feasible genotyping methods that usually have to be developed by the user.

Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

BACKGROUND: Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i)-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. RESULTS: Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i)-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. CONCLUSIONS: The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis.

Poly(ADP-ribose) polymerases PARP1 and PARP2 modulate topoisomerase II beta (TOP2B) function during chromatin condensation in mouse spermiogenesis.

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.