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

Palabras clave extraídas del título[EN] 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/cm25 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 qualitySIThis work was supported by the Spanish Ministry of Economy and Competitiveness (project AGL2011-27787

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

[EN] 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 environmentSIThis work was supported by Ministerio de Economı́a y Competitividad (AGL2011- 27787 and AGL2014-53167

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

[EN] 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 damageSISpanish Ministry of Economy and Competitiveness (project AGL2011-27787; AGL2014-53167-C3-3-R), Junta de Castilla y León (Spain) (EDU/1083/2013) and the Fondo Social Europe

Genetic and epigenetic alterations induced by bisphenol A exposure during different periods of spermatogenesis: from spermatozoa to the progeny

Exposure to bisphenol A (BPA) has been related to male reproductive disorders. Since this endocrine disruptor also displays genotoxic and epigenotoxic effects, it likely alters the spermatogenesis, a process in which both hormones and chromatin remodeling play crucial roles. The hypothesis of this work is that BPA impairs early embryo development by modifying the spermatic genetic and epigenetic information. Zebrafish males were exposed to 100 and 2000 μg/L BPA during early spermatogenesis and during the whole process. Genotoxic and epigenotoxic effects on spermatozoa (comet assay and immunocytochemistry) as well as progeny development (mortality, DNA repairing activity, apoptosis and epigenetic profile) were evaluated. Exposure to 100 µg/L BPA during mitosis slightly increased sperm chromatin fragmentation, enhancing DNA repairing activity in embryos. The rest of treatments promoted high levels of sperm DNA damage, triggering apoptosis in early embryo and severely impairing survival. Regarding epigenetics, histone acetylation (H3K9Ac and H3K27Ac) was similarly enhanced in spermatozoa and embryos from males exposed to all the treatments. Therefore, BPA male exposure jeopardizes embryonic survival and development due to the transmission of a paternal damaged genome and of a hyper-acetylated histone profile, both alterations depending on the dose of the toxicant and the temporal window of exposureS

Male exposure to bisphenol a impairs spermatogenesis and triggers histone hyperacetylation in zebrafish testes

[EN] Bisphenol A (BPA) is an endocrine disruptor whose ubiquitous presence in the environment has been related with impairment of male reproduction. BPA can cause both transcriptomic and epigenetic changes during spermatogenesis. To evaluate the potential effects of male exposure to BPA, adult zebrafish males were exposed during spermatogenesis to doses of 100 and 2000 μg/L, which were reported in contaminated water bodies and higher than those allowed for human consumption. Fertilization capacity and survival at hatching were analysed after mating with untreated females. Spermatogenic progress was analysed through a morphometrical study of testes and apoptosis was evaluated by TUNEL assay. Testicular gene expression was evaluated by RT-qPCR and epigenetics by using ELISA and immunocytochemistry. In vitro studies were performed to investigate the role of Gper. Chromatin fragmentation and the presence of transcripts were also evaluated in ejaculated sperm. Results on testes from males treated with the highest dose showed a significant decrease in spermatocytes, an increase in apoptosis, a downregulation of ccnb1 and sycp3, all of which point to an alteration of spermatogenesis and to meiotic arrest and an upregulation of gper1 and esrrga receptors. Additionally, BPA at 2000 μg/L caused missregulation of epigenetic remodelling enzymes transcripts in testes and promoted DNA hypermethylation and H3K27me3 demethylation. BPA also triggered an increase in histone acetyltransferase activity, which led to hyperacetylation of histones (H3K9ac, H3K14ac, H4K12ac). In vitro reversion of histone acetylation changes using a specific GPER antagonist, G-36, suggested this receptor as mediator of histone hyperacetylation. Males treated with the lower dose only showed an increase in some histone acetylation marks (H3K14ac, H4K12ac) but their progeny displayed very limited survival at hatching, revealing the deleterious effects of unbalanced paternal epigenetic information. Furthermore, the highest dose of BPA led to chromatin fragmentation, promoting direct reproductive effects, which are incompatible with embryo developmentSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (project AGL2014-53167-C3-3-R), Junta de Castilla y León (Spain) (EDU/1083/2013), the Fondo Social Europeo and by an EMBO Short-Term Fellowship to SG

Cardiogenesis impairment promoted by bisphenol A exposure is successfully counteracted by epigallocatechin gallate

[EN] Exposure to the emerging contaminant bisphenol A (BPA) is ubiquitous and associated with cardiovascular disorders. BPA effect as endocrine disruptor is widely known but other mechanisms underlying heart disease, such as epigenetic modifications, remain still unclear. A compound of green tea, epigallocatechin gallate (EGCG), may act both as anti-estrogen and as inhibitor of some epigenetic enzymes. The aims of this study were to analyze the molecular processes related to BPA impairment of heart development and to prove the potential ability of EGCG to neutralize the toxic effects caused by BPA on cardiac health. Zebrafish embryos were exposed to 2000 and 4000 μg/L BPA and treated with 50 and 100 μM EGCG. Heart malformations were assessed at histological level and by confocal imaging. Expression of genes involved in cardiac development, estrogen receptors and epigenetic enzymes was analyzed by qPCR whereas epigenetic modifications were evaluated by whole mount immunostaining. BPA embryonic exposure led to changes in cardiac phenotype, induced an overexpression of hand2, a crucial factor for cardiomyocyte differentiation, increased the expression of estrogen receptor (esr2b), promoted an overexpression of a histone acetyltransferase (kat6a) and also caused an increase in histone acetylation, both mechanisms being able to act in sinergy. EGCG treatment neutralized all the molecular alterations caused by BPA, allowing the embryos to go on with a proper heart development. Both molecular mechanisms of BPA action (estrogenic and epigenetic) likely lying behind cardiogenesis impairment were successfully counteracted by EGCG treatmentSIThis work was funded by the Spanish Ministry of Economy and Competitiveness (Project AGL2014-53167-C3-3-R; PhD Grant BES-2015-071885)

Differential Gene Susceptibility to Sperm DNA Damage: Analysis of Developmental Key Genes in Trout

Palabras clave extraídas de la publicación[EN] 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)SIThis work was supported by the Junta de Castilla y León (Spain) (project LE365A11-2 and EDU/828/2014), the Spanish Ministry of Economy and Competitiveness (project AGL2011-27787) and Fondo Social Europeo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Cp*Fe(Me2PCH2CH2PMe2)(CHO) : hydride shuttle reactivity of a thermally stable formyl complex.

[Cp*Fe(Me2PCH2CH2PMe2)(CO)]+ [BArF24]− has been synthesised and characterised using single crystal X-ray diffraction, NMR and IR spectroscopies. Reduction of the CO ligand using Na[Et3BH] produces the corresponding neutral formyl complex Cp*Fe(Me2PCH2CH2PMe2)(CHO), that is very thermally stable, and which is attributed to the electron-releasing properties of the spectator ligands. This compound is a potent hydride donor which exists in equilibrium with [Et3BH]−, Et3B, and the structural isomer (η4-C5Me5H)Cp*Fe(Me2PCH2CH2PMe2)(CO), resulting from reversible hydride migration to the Cp* ligand

Consumo de alimentos, composición corporal y rendimiento físico en hombres y mujeres jóvenes judadores de fútbol

Introduction: Few studies have analysed the nutritional habits, body composition and physical condition of young soccer players among different categories and further differentiating between males and females. Therefore, the aim was to analyze differences in food consumption habits, body composition parameters and physical performance among young soccer players. Material and Methods: A cross-sectional study was performed during the 2019-2020 competitive season. Forty-eight soccer players (16-18 years) were classified by age and sex into three groups: under 16 and under 18 years males (U16 and U18), and under 16 years females (U16 female). Food habits, anthropometry, body composition and physical performance variables were collected. A one-way analysis of variance (ANOVA) with least significant difference post hoc comparison (Bonferroni correction) was used. Results: We found that U16 females consumed lower pork and bread and higher mollusk and shellfish than males (p<0.05). U16 males consumed less fruits (p<0.05) and more olive oil (p<0.05) than U16 females and had lower muscle mass in lower limbs than U18 male (p<0.05, ES=0.80-0.96); males showed higher muscle mass in lower limbs (p<0.01, ES=1.80-2.59) and lower %fat than females (p<0.01, ES=1.73); and U18 males presented higher weight than females (p<0.01, ES=1.64). U18 males showed better performances (p<0.05-0.01, ES=0.90-1.38) in maximal sprint trials (40-m length), counter movements jumps (CMJ) and CMJ with dominant leg, maximal bilateral horizontal jumps (HJ), HJ with dominant and non-dominant leg than U16 males. Males showed better results in linear straight sprint test (LSST, p<0.05; ES=1.66-5.16), change of direction ability (CODA, p<0.05; ES=2.82-5.46) and jumping (p<0.05; ES=1.60-4.12) performances. Conclusion: In conclusion, sex- and age- specific differences were detected in the healthier dietary practices and body composition parameter in youth of soccer players. Also, better physical performances were found as age increases, except for CODA, and males exhibited higher performances in LSST, CODA and jumping than females.Introducción: Pocos estudios han sido encontrados donde se analicen los hábitos nutricionales, la composición corporal y la condición física de jóvenes futbolistas entre diferentes categorías y diferenciando entre chicos y chicas. Por tanto, el objetivo fue analizar las diferencias en los hábitos de consumo de alimentos, los parámetros de composición corporal y el rendimiento físico en jóvenes futbolistas. Material y métodos: Se realizó un estudio transversal durante la temporada competitiva 2019-2020. Cuarenta y ocho futbolistas (16-18 años) fueron clasificados por edad y sexo en tres grupos: menores de 16 y menores de 18 años masculinos (U16 y U18), y menores de 16 años féminas (U16 femenino). Se recogieron variables de hábitos alimentarios, antropometría, composición corporal y rendimiento físico. Se utilizó un análisis de varianza de (ANOVA) de un factor con post hoc de Bonferroni. Resultados: Observamos que las chicas U16 consumieron menos carne de cerdo y pan y más moluscos y mariscos que los chicos (p<0,05). Los chicos U16 consumieron menos frutas (p<0,05) y más aceite de oliva (p<0,05) que las chicas U16 y tuvieron menor masa muscular en miembros inferiores que los chicos U18 (p <0,05, ES=0,80-0,96); los chicos mostraron mayor masa muscular en miembros inferiores (p <0,01, ES=1,80-2,59) y menor porcentaje de grasa que las chicas (p<0,01, ES=1,73); y los chicos U18 presentaron mayor peso que las chicas (p<0,01, ES=1,64). Los chicos U18 mostraron mejores rendimientos (p<0,05-0,01, ES=0,90-1,38) en pruebas de sprint máximo (40 m de longitud), salto en contramovimiento (CMJ) y CMJ con pierna dominante, saltos horizontales bilaterales máximos (HJ), HJ con pierna dominante y no dominante que los chicos U16. Los chicos mostraron mejores resultados en los esprints (LSST, p<0,05; ES=1,66-5,16), el cambio de dirección (CODA, p<0,05; ES = 2,82-5,46) y los saltos (p<0,05; ES=1,60-4,12). Conclusión: En conclusión, se observaron diferencias por sexo y edad en las prácticas dietéticas y los parámetros de composición corporal en los jóvenes futbolistas. Además, mejor rendimiento físico a medida que aumentaba la edad, excepto para el CODA. Los chicos exhibieron mejores rendimientos en LSST, CODA y saltos que las chicas

La aplicación de flipped classroom en la docencia de biología celular en el primer grado de ciencias ambientales

La metodología flipped classroom se basa en que los estudiantes realicen parte del proceso de aprendizaje previamente a las clases presenciales. Se pretende que la participación de los estudiantes sea más intensa, y que la clase presencial pueda utilizarse más eficientemente. Biología Fundamental es una asignatura Básica de 1er curso del Grado en CC. Ambientales de la Universidad de León. Incluye aspectos básicos de bioquímica, biología celular y genética. El Área de Biología Celular participa con 14 h de docencia magistral y 8 h de práctica (noviembre), con un examen al finalizar (test y preguntas cortas de razonamiento). Es poco atractiva para muchos estudiantes, ya que sus contenidos no se corresponden con las expectativas que tienen del Grado, y además éstos tienen procedencias muy diversas, con lo que sus conocimientos previos no siempre son los apropiados para la asignatura. El presente trabajo describe una experiencia de dos cursos consecutivos correspondiente a la parte impartida por Biología Celular (las calificaciones se dan sobre 10, como mediana±desviación absoluta de la mediana —MAD—, unos estadísticos más robustos que la media±SD). En el curso 2014/15 (91 alumnos), se utilizó una metodología "clásica", consistente en clases magistrales y prácticas sin actividades previas. En el curso 2015/16 (86 alumnos), la metodología docente se inspiró en el flipped learning. Se modificó todo el material docente del campus virtual y se pidió a los estudiantes que, previamente a las clases (prácticas y magistrales), revisasen el material y que realizasen unas pruebas tipo test (con cierto peso en la evaluación). La percepción subjetiva fue de un ligero incremento de la atención y la comprensión. Los aprobados (prácticas y examen) se incrementó de un 49,4% en 2014 a un 74,3% en 2015. Las calificaciones se incrementaron 1 MAD, de 4,8±1,2 a un 6,0±1,6 (sólo aprobados, de 5,6±0,5 a 6,4±0,8; cuantil 95%: 6,7 a 7,9). No obstante, hay aspectos que deben ser mejorados considerablemente. Aunque las preguntas de razonamiento también incrementaron su calificación, éstas son aún muy bajas (1,2±1,9 a 2,5±1,9). Los resultados han sido positivos. Sólo se han utilizado los aspectos más básicos de la metodología. Hemos conseguido mejorar los resultados, pero sólo en las operaciones intelectuales más inferiores (taxonomía de Bloom), evaluadas principalmente por los tests. En sucesivos cursos pretendemos introducir otros elementos para incrementar el aprendizaje significativo