Paternal DNA Damage Research Articles

Mutations in the germline: How the mother repairs the father’s damaged genome

Germline mutations can have a severe impact on genetic diseases, genome evolution and the fate of a species. The vast majority of inheritable mutations are passed on by the paternal genome. We discovered how paternal DNA damage is repaired by maternal repair leading to the inheritance of structural variants.

Inheritance of paternal DNA damage by histone-mediated repair restriction

How paternal exposure to ionizing radiation affects genetic inheritance and disease risk in the offspring has been a long-standing question in radiation biology. In humans, nearly 80% of transmitted mutations arise in the paternal germline1, but the transgenerational effects of ionizing radiation exposure has remained controversial and the mechanisms are unknown. Here we show that in sex-separated Caenorhabditis elegans strains, paternal, but not maternal, exposure to ionizing radiation leads to transgenerational embryonic lethality. The offspring of irradiated males displayed various genome instability phenotypes, including DNA fragmentation, chromosomal rearrangement and aneuploidy. Paternal DNA double strand breaks were repaired by maternally provided error-prone polymerase theta-mediated end joining. Mechanistically, we show that depletion of an orthologue of human histone H1.0, HIS-24, or the heterochromatin protein HPL-1, could significantly reverse the transgenerational embryonic lethality. Removal of HIS-24 or HPL-1 reduced histone 3 lysine 9 dimethylation and enabled error-free homologous recombination repair in the germline of the F1 generation from ionizing radiation-treated P0 males, consequently improving the viability of the F2 generation. This work establishes the mechanistic underpinnings of the heritable consequences of paternal radiation exposure on the health of offspring, which may lead to congenital disorders and cancer in humans.

The Capacity to Repair Sperm DNA Damage in Zygotes is Enhanced by Inhibiting WIP1 Activity.

Maintaining genome integrity in germ cells is essential not only for successful fertilization and embryo development, but also to ensure proper transmission of genetic information across generations. However, unlike oocytes, sperm are incapable of repairing DNA damage. Therefore, sperm DNA damage is repaired after fertilization in zygotes using maternal DNA repair factors. In this study, we found that zygotic repair of paternal DNA damage is enhanced by inhibiting WIP1 activity. Oxidative stress induced DNA damage in sperm and severely impaired motility. Although DNA damage in sperm did not compromise fertilization, it increased DNA damage in the paternal pronucleus of zygotes. However, WIP1 inhibition during fertilization reduced DNA damage in the paternal pronucleus, improving the rate of two-cell development, and subsequent zygotic genome activation. Therefore, our results suggest that WIP1 inhibition could enhance maternal DNA repair capacity and thereby decrease paternal DNA damage in zygotes.

O-210 Sperm DNA fragmentation (SDF) is not associated with adverse maternal and neonatal outcomes in IVF-ICSI cycles with autologous oocytes

Abstract Study question Does an elevated SDF (>15%) increase the odds of adverse maternal and neonatal outcomes in autologous oocyte IVF-ICSI cycles from unselected couples? Summary answer No adverse effects of high SDF on obstetric and neonatal outcomes have been found in couples with sperm fragmentation undergoing IVF-ICSI cycles with own eggs. What is known already Sperm chromatin integrity assessment has been implemented as an additional tool in the clinical evaluation of sperm quality in infertile patients undergoing an assisted reproduction treatment. All of the published reports to date appraise its effect on clinical outcomes, and how it impacts embryo quality and the pregnancy chances after IVF and ICSI cycles. Sperm DNA integrity has also been hypothesized to affect offspring health but not many studies have reported in humans if an elevated SDF raises the risks of obstetric, delivery and neonatal outcomes. Study design, size, duration Multicentric retrospective cohort study of all IVF-ICSI cycles using autologous oocytes between January 2000-March 2019 at Spain IVIRMA clinics of couples with a SDF test on their ejaculated semen. The sperm fragmentation index was measured in all men with TUNEL assay. The database included 228 couples which had a delivery with at least a newborn. Subjects were divided into two study groups according to their level of SDF: ≤15% (low SDF) or > 15% (high SDF). Participants/materials, setting, methods Patients with missed information on maternal and neonatal outcomes were not counted for the analysis. The obstetric outcomes were gestational age, gestational diabetes, preeclampsia (hypertension with proteinuria after 20 weeks of gestation) and type of delivery. Neonatal outcomes were sex, birth weight, length, head circumference, Apgar score at 1, 5, 10 minutes, and neonatal intensive care unit (NICU) admission. Student’s t-test and Fisher’s test were used for statistical analysis. A p-value<0.05 was considered statistically significant. Main results and the role of chance Maternal age mean was 37.4 years (95%CI 36.9-38.0) in ≤ 15%SDF group and 37.2 years (95%CI 36.1-38.4) in > 15%SDF group (p = 0.8). Similar gestational age was found, 41.8 weeks (95%CI 41.3-42.2) in ≤ 15%SDF and 41.3 weeks (95%CI 40.4-42.3) in > 15%SDF. Gestational diabetes incidence was higher in > 15%SDF compared to ≤ 15%SDF group (3.5% versus 1.7% (OR = 2.0 (95%CI 0.03-39.8), p = 0.5). Equally, the incidence of preeclampsia was 3.6% in patients with high SDF versus 1.7% in couples with low SDF, OR = 2.1 (95%CI 0.03-41.3), p = 0.5. Type of delivery frequency was in the ≤15%SDF group 61.9% vaginal and 38.1% cesarean, while in the >15%SDF group 62.1% vaginal and 37.9% cesarean (OR = 1.0 (95%CI 0.4-2.6), p = 1.0). The overall proportion of singleton pregnancies was 87.2% (95%CI 82.4-91.2) and twins 12.8% (95%CI 8.8-17.6). There were no statistically differences between groups in the rate of delivery of twins and in the sex ratio of the newborns. When comparing the newborns of ≤ 15%SDF with >15%SDF group, the average of weight was 3011.7g (95%CI 2912.2-3111.2) versus 2986.4g (95%CI 2753.1-3219.7), of length was 49.2cm (95%CI 48.3-50.0) versus 49.5cm (95%CI 49.2-49.9), of head circumference was 34.9cm (95%CI 34.6-35.2) versus 34.3cm (95%CI 33.4-35.2). No statistically differences were observed for Apgar punctuation and for NICU admission. Limitations, reasons for caution Due to the retrospective nature of the study we have missing data from the lack of follow-up of many patients after the confirmation of the ongoing pregnancy. Although pregnancies of couples with elevated SDF have a higher incidence of gestational diabetes and preeclampsia, the sample size evaluated is a limitation. Wider implications of the findings This is one of the first reports to evaluate the relationship between paternal DNA damage and obstetric risks and neonatal health in couples with high SDF who underwent IVF-ICSI in our centers. Despite SDF did not jeopardize the maternal and neonatal outcomes, more studies are needed to confirm this conclusion. Trial registration number NA

Effect of Paternal DNA Damage on Paternal DNA Degradation and Early Embryonic Development in Mouse Embryo: Supporting Evidence by GammaH2AX Expression

Effect of Paternal DNA Damage on Paternal DNA Degradation and Early Embryonic Development in Mouse Embryo: Supporting Evidence by GammaH2AX Expression

Tolerance to paternal genotoxic damage promotes survival during embryo development in zebrafish (Danio rerio).

ABSTRACTSpermatozoa carry DNA damage that must be repaired by the oocyte machinery upon fertilization. Different strategies could be adopted by different vertebrates to face the paternal genotoxic damage. Mammals have strong sperm selection mechanisms and activate a zygotic DNA damage response (DDR) (including cell cycle arrest, DNA repair and alternative apoptosis) in order to guarantee the genomic conformity of the reduced progeny. However, external fertilizers, with different reproductive strategies, seem to proceed distinctively. Previous results from our group showed a downregulation of apoptotic activity in trout embryos with a defective DNA repairing ability, suggesting that mechanisms of tolerance to damaged DNA could be activated in fish to maintain cell survival and to progress with development. In this work, zebrafish embryos were obtained from control or UV-irradiated sperm (carrying more than 10% of fragmented DNA but still preserving fertilization ability). DNA repair (γH2AX and 53BP1 foci), apoptotic activity, expression of genes related to DDR and malformation rates were analyzed throughout development. Results showed in the progeny from damaged sperm, an enhanced repairing activity at the mid-blastula transition stage that returned to its basal level at later stages, rendering at hatching a very high rate of multimalformed larvae. The study of transcriptional and post-translational activity of tp53 (ZDF-GENE-990415-270) revealed the activation of an intense DDR in those progenies. However, the downstream pro-apoptotic factor noxa (ZDF-GENE-070119-3) showed a significant downregulation, whereas the anti-apoptotic gene bcl2 (ZDF-GENE-051015-1) was upregulated, triggering a repressive apoptotic scenario in spite of a clear genomic instability. This repression can be explained by the observed upregulation of p53 isoform Δ113p53, which is known to enhance bcl2 transcription. Our results showed that tp53 is involved in DNA damage tolerance (DDT) pathways, allowing the embryo survival regardless of the paternal DNA damage. DDT could be an evolutionary mechanism in fish: tolerance to unrepaired sperm DNA could introduce new mutations, some of them potentially advantageous to face a changing environment.

Zygotic chromosomal structural aberrations after paternal drug treatment.

In recent years, the field of male-mediated reproductive toxicology has received growing attention. It is now well-established that many drugs, chemicals, and environmental factors can harm male germ cells by inducing DNA damage. Male germ cells have extensive repair mechanisms that allow detection and repair of damaged DNA during the early phases of spermatogenesis. However, during the later phase of spermiogenesis, when the haploid spermatids undergo chromatin condensation and become transcriptionally quiescent, their ability to repair damaged DNA is lost.12 It is also thought that the highly compacted chromatin of the sperm can protect DNA against damage.3 Therefore, it is expected that late spermatids will be most susceptible to DNA damaging agents. Unrepaired or misrepaired damage in the germ cells leads to the generation of spermatozoa with DNA damage that can be transmitted to the next generation. Fortunately, the maternal DNA repair machinery is capable of recognizing and repairing, at least to some degree, damaged paternal DNA after fertilization in the zygote. Therefore, the efficiency of the maternal repair machinery will greatly influence the risk of transmitting paternal DNA damage to offspring.4

Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout.

Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins. Differential packaging has been related to early or late transcription and also to differential susceptibility to genotoxic damage. Genes located in the more accessible HDNA could be more susceptible to injuries than those located in PDNA, being potential biomarkers of paternal DNA damage. Fish sperm chromatin organization is much diversified, some species lacking protamines and some others totally depleted of histones. Analyzing genotoxic damage in a species homogeneously compacted with some sperm nuclear basic protein type, could help in deciphering the clues of differential susceptibility to damage. In the present study we analyzed in rainbow trout the differential susceptibility of nine genes to UV irradiation and H2O2 treatment. The absence of histones in the sperm nuclei was confirmed by Western blot. The chromatin fractionation in sensitive and resistant regions to PvuII (presumably HDNA-like and PDNA-like, respectively) revealed that the nine genes locate in the same resistant region. The number of lesions promoted was quantified using a qPCR approach. Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy. UV irradiation promoted similar number of lesions in all the analyzed genes and a homogenous distribution of 8-OHdG within the nuclei. 8-OHdG was located in the peripheral area of the nucleus after H2O2 treatment, which promoted a significantly higher number of lesions in developmental-related genes (8.76–10.95 lesions/10 kb) than in rDNA genes (1.05–1.67 lesions/10 kb). We showed for the first time, that differential susceptibility to damage is dependent on the genotoxic mechanism and relies on positional differences between genes. Sensitive genes were also analyzed in cryopreserved sperm showing a lower number of lesions than the previous treatments and a predominant peripheral distribution of oxidative damage (8-OHdG).

Mouse Zygotes Respond to Severe Sperm DNA Damage by Delaying Paternal DNA Replication and Embryonic Development

Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

Paternal DNA Damage Resulting From Various Sperm Treatments Persists After Fertilization and Is Similar Before and After DNA Replication

In spite of its highly condensed state, sperm DNA is vulnerable to damage that can originate from oxidative stress, the activity of sperm-specific nucleases, or both. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes, and during this extensive remodeling, it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization and before or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation, spermatozoa from 3 groups were taken for comet assay, or for intracytoplasmic sperm injection into prometaphase I oocytes to visualize prematurely condensed single-chromatid chromosomes, or into mature metaphase II oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared with control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated, but not with fresh, sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates before and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage-inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to 2 treatments was ameliorated in the fertilized oocytes, suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis and took place during oocyte maturation.

Involvement of inducible nitric oxide synthase in DNA fragmentation in various testicular germ cells of ethanol-treated rats

Chronic alcohol intake is associated with testicular damage, hypogonadism and infertility. Previously, we reported enhancement of testicular germ cell apoptosis in ethanol-treated rats (ETR) associated with upregulation of apoptosis related genes. Fas, FasL, p53, and activation of caspases. Here, we investigated the involvement of inducible nitric oxide synthase (iNOS) which is a major mediator of oxidative stress in ethanol-induced germ cell apoptosis. Moreover, we explored the various germ cell types undergoing apoptotic DNA fragmentation and their relationship to phagocytosing Sertoli cells. Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. iNOS expression in control and ETR was investigated using immunohistochemistry. DNA fragmentation in various germ cells of ETR was detected using TUNEL method under both light and electron microscopy. Damaged spermatids with apoptotic features were observed by transmission electron microscopy (TEM). Compared to very weak expression of iNOS in control testis, marked increase of iNOS was detected in Sertoli, germ cells and some interstitial cells in ETR. Phagocytosed retained elongated spermatids undergoing DNA fragmentation was detected by TUNEL method in ETR, in addition to spermatids undergoing nuclear vacuolization and chromatin degeneration using TEM. The upregulation of iNOS in the testes of ETR may induce apoptosis of germ cells through the generation of excessive nitric oxide and androgen suppression. Increased DNA fragmentation in spermatids of ETR may be involved in infertility and offspring diseases. We recommend for further research to detect and treat oxidative stress-mediated paternal DNA damage in alcoholics.

Reproductive Consequences of Paternal Genotoxin Exposure in Marine Invertebrates

Chemicals with the potential to damage DNA are increasingly present in the marine environment; yet our understanding of the long-term consequences of DNA damage for populations remains limited. We explore the impact of paternal genotoxin exposure on the reproductive biology of two ecologically important free-spawning marine invertebrates: the polychaete Arenicola marina and the mussel Mytilus edulis. Males were exposed in vivo for 72 h to methyl methanesulfonate and benzo(a)pyrene and the impact on somatic cells and sperm assessed using the Comet assay. A strong correlation between DNA damage in somatic cells and sperm was observed after 24 h exposure (P < 0.001). Recovery in sperm was significantly lower than in coelomocytes after 72 h. The fertilization success of DNA-damaged sperm was unaffected, but a significant percentage of embryos derived from sperm with induced DNA damage exhibited severe developmental abnormalities within 24 h of fertilization with potential long-term consequences for population success. Further research is required to determine the mechanism by which paternal DNA damage causes disruption of development at this early stage.

Paternal DNA Damage Resulting from Various Sperm Treatments can be Detected both Before and after Fertilization and is Similar prior and after DNA Synthesis.

Paternal DNA Damage Resulting from Various Sperm Treatments can be Detected both Before and after Fertilization and is Similar prior and after DNA Synthesis.