Cristina Fernández-Díez

Transgenerational inheritance of heart disorders caused by paternal bisphenol A exposure

Bisphenol A (BPA) is an endocrine disruptor used in manufacturing of plastic devices, resulting in an ubiquitous presence in the environment linked to human infertility, obesity or cardiovascular diseases. Both transcriptome and epigenome modifications lie behind these disorders that might be inherited transgenerationally when affecting germline. To assess potential effects of paternal exposure on offspring development, adult zebrafish males were exposed to BPA during spermatogenesis and mated with non-treated females. Results showed an increase in the rate of heart failures of progeny up to the F2, as well as downregulation of 5 genes involved in cardiac development in F1 embryos. Moreover, BPA causes a decrease in F0 and F1 sperm remnant mRNAs related to early development. Results reveal a paternal inheritance of changes in the insulin signaling pathway due to downregulation of insulin receptor β mRNAs, suggesting a link between BPA male exposure and disruption of cardiogenesis in forthcoming generations.

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).

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss)

Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61 657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depth.

Impact of sperm DNA damage and oocyte-repairing capacity on trout development

Zygotic repair of paternal DNA is essential during embryo development. In spite of the interest devoted to sperm DNA damage, its combined effect with defect-repairing oocytes has not been analyzed. Modification of the breeding season is a common practice in aquaculture. This practice reduces developmental success and could affect the both factors: sperm DNA integrity and oocyte repair capacity. To evaluate the maternal role, we analyzed the progeny outcome after fertilizing in-season trout oocytes with untreated and with UV-irradiated sperm. We also analyzed the offspring obtained out of season with untreated sperm. The analysis of the number of lesions in 4 sperm nuclear genes revealed an increase of 1.22-11.18 lesions/10 kb in out-of-season sperm, similar to that obtained after sperm UV irradiation (400 µW/cm(2)5 min). Gene expression showed in out-of-season oocytes the overexpression of repair genes (ogg1, ung, lig3, rad1) and downregulation of tp53, indicating an enhanced repairing activity and reduced capacity to arrest development upon damage. The analysis of the progeny in out-of-season embryos revealed a similar profile tolerant to DNA damage, leading to a much lower apoptotic activity at organogenesis, lower hatching rates and increased rate of malformations. The effects were milder in descendants from in-season-irradiated sperm, showing an enhanced repairing activity at epibolia. Results point out the importance of the repairing machinery provided by the oocyte and show how susceptible it is to environmental changes. Transcripts related to DNA damage signalization and repair could be used as markers of oocyte quality.

Distribution of DNA damage in the sperm nucleus: A study of zebrafish as a model of histone-packaged chromatin

Reproductive defects can occur when the integrity of the male gamete genome is affected. Sperm chromatin is not homogeneous, having relaxed regions which are more accessible to the transcription machinery in the embryo, and thought to be specially sensitive to DNA damage. The level of damage in specific genes located in these sensitive regions could represent an early biomarker of damage. Our objective is to test the hypothesis that these more relaxed regions show greater susceptibility to damage in zebrafish, a species lacking protamines and whose sperm chromatin is compacted with histones. After sperm UV irradiation, treatment with H2O2 and cryopreservation, global chromatin fragmentation was evaluated using the TUNEL assay, and the number of lesions per 10 Kb in specific genes (hoxa3a, hoxb5b, sox2, accessible for early transcription and rDNA 18S and rDNA 28S) was quantified by using a qPCR approach. Additionally, oxidative damage within the sperm nucleus and the potential colocalization of this injury with histone H3 and TOPO IIα+β were located by using immunofluorescence. UV irradiation produced the highest degree of fragmentation (p = 0.041) and the highest number of lesions per 10 Kb in all the genes, but no differences were observed in sensitivity to damage in the studied genes (ranging from 14.93 to 8.03 lesions per 10 Kb in hoxb5b and 28S, respectively). In contrast, H2O2 and cryopreservation caused varying levels of damage in the analyzed genes which was not related to their accessibility, ranging from 0.00 to 1.65 lesions per 10 Kb in 28S and hoxb5b, respectively, after H2O2 treatment, and from 0.073 to 5.51 in 28S and sox2, respectively, after cryopreservation. Immunodetection near oxidative lesions also revealed different spatial patterns depending on the treatments used, these being mostly homogeneous with UV irradiation or cryopreservation, and peripherally located around the nucleus after H2O2 treatment. Oxidative lesions did not colocalize with histone H3 or TOPO IIα+β, thus demonstrating that the relaxed DNA regions associated with these proteins were not more vulnerable to oxidative damage. Results suggest that accessibility of each agent to the nucleus could be the main factor responsible for the distribution of sperm DNA damage rather than the organization of the chromatin. Lesions in these genes important to early embryo development assayed in this study cannot be used as biomarkers of global DNA damage.

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

ABSTRACT Spermatozoa 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. Summary: In fish embryos, genomic instability generated by fertilization with DNA damaged sperm activates mechanisms of DNA damage tolerance, which seems to be mediated by Δ113p53 expression, promoting survival.