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Our current results suggest that the application of HDACi in combination with drugs that target DNMT may represent a promising option for the treatment of medulloblastoma.

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Background: Benign prostatic hyperplasia (BPH) is one of the most common reproductive disorders in both male dogs and men. Finasteride, a synthetic inhibitor of the enzyme 5α-reductase, is widely used as medical treatment. Although sperm can be affected by both BPH and finasteride treatment, the direct influence on DNA integrity remains unclear. Thus, the aim of this study was to verify the direct effect of BPH and/or finasteride treatment on DNA integrity of dog spermatozoa. A 2 × 2 factorial experiment was designed with 20 male dogs assigned to 4 experimental groups: BPH Group (n = 5), BPH-Finasteride Group (n = 5), Non-BPH Finasteride-Treated Group (n = 5) and Non-BPH Untreated Group (n = 5). Sperm evaluation was performed monthly for 60 days after the start of finasteride therapy or BPH diagnosis (D0, D30 and D60). Sperm DNA integrity was analyzed through fragmentation susceptibility (toluidine blue staining and Sperm Chromatic Structure Assay-SCSA), direct evaluation of DNA fragmentation (Sperm Chromatin Dispersion Assay-SCDA) and sperm protamination (chromomycin A3). Results: Sperm DNA integrity was not affected by finasteride treatment. However, BPH dogs had higher susceptibility to sperm DNA acid denaturation (SCSA) compared to dogs not presenting BPH, as well as lower percentage of sperm with DNA integrity (toluidine blue staining). Conclusion: In conclusion, benign prostatic hyperplasia causes post-testicular sperm DNA damage, albeit finasteride treatment itself does not directly influence sperm DNA integrity.

Belgian Blue bulls are more susceptible to high temperature and humidity index (THI) than most other cattle breeds. Here, we investigated whether high ambient temperature during summer affected semen quality and subsequent embryo development in Belgian Blue cattle. For this purpose, semen samples were collected from six healthy mature Belgian Blue bulls in March (Low THI group; THI between 30.6 and 56.4) and August 2016 (High THI group; maximum THI of 83.7 during meiotic and spermiogenic stages of spermatogenesis; 14–28 days prior to semen collection) respectively. Motility, morphology, acrosome integrity, chromatin condensation, viability, and reactive oxygen species production were assessed for frozen-thawed semen. Moreover, the efficiency of blastocyst production from the frozen-thawed semen samples of the two groups was determined in vitro . Blastocyst quality was determined by assessing inner cell mass ratio and apoptotic cell ratio. Fresh ejaculates showed a higher sperm concentration in low THI when compared to the high THI group ( P ≤ 0.05), whereas semen volume, subjective motility, and total sperm output were not affected ( P > 0.05). In frozen-thawed semen, total and progressive motility, viability, and straight-line velocity were lower in high THI compared to the low THI group ( P < 0.05), while H 2 O 2 concentration, aberrant chromatin condensation, and abnormal spermatozoa were higher in the high THI group ( P < 0.05). Blastocyst rates were significantly higher when low THI samples were used ( P < 0.05). Moreover, the total cell number and trophectoderm cells were significantly higher ( P < 0.05) in blastocysts derived from low THI samples, whereas the apoptotic cell ratio was significantly higher ( P < 0.01) in blastocysts derived from high THI spermatozoa. In summary, our data show that elevated ambient temperature and humidity during summer can decrease the quality of frozen-thawed spermatozoa in Belgian Blue bulls and also affect subsequent embryo development.

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Concerns about global climate change reducing animal fertility are arising. Therefore, our objective was to determine the effects of increased environmental temperature on Holstein bulls and its effects on sperm quality and embryo development. Frozen semen samples were obtained from 6 bulls exposed to natural heat stress (HS) in August 2016, compared with a lower temperature (control) in March 2016 (temperature-humidity index of up to 74.5 and 40.6, respectively). We evaluated sperm morphology, embryo development, gene expression, inner cell mass/trophectoderm ratio (ICM/TE), and apoptosis cell ratio of Day-8 blastocysts. Sperm morphology was evaluated using eosin/nigrosin staining. Blastocysts were produced by routine in vitro methods (Wydooghe et al. 2014 Reprod. Fertil. Dev.). Cleavage rates were determined 48h after insemination, and blastocyst rates were determined on Days 7 and 8. Expression of NANOG, SOX2, POU5F1, DNMT (1, 3a, 3b), HSP (A1a, A2, A8, 10, 60, 90), HSF1, IFNT2, H19, SNRPN, IGF2, IGF2R, MEST, PHLDA2, MEG3, MEG9, PEG10 and PLAGL1 were analysed. Total RNA was extracted from Day-8 blastocysts for gene expression analysis using RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Reverse transcription and qPCR were performed with iScript (BioRad, Hercules, CA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (BioRad) in a CFX Connect system (BioRad). Blastocysts were differentially stained (Wydooghe et al. 2011 Anal. Biochem.) and analysed using a Leica TCS-SP8×confocal microscope (Leica Microsystems, Wetzlar, Germany). Data analyses included a GLM procedure and paired-samples Student’s t test (P ≤ 0.05). A normal distribution was verified with a UNIVARIATE procedure and Shapiro-Wilk test. A Wilcoxon signed rank test was used to analyse qPCR data. Detached heads (P=0.006) and coiled tails (P=0.018) were significantly lower in the HS group (4.9 and 0.2%, respectively) compared with the control (5.5 and 0.5%). Moreover, proximal droplets (P=0.051) were lower in the HS group (0.7%) compared with the control (1.3%). Remarkably, cleavage and blastocyst rates at Days 7 and 8 were significantly higher in control (78.4, 19.6 and 29.5%, respectively) compared with HS (75, 14.5 and 23.2%). Early and normal blastocysts were grouped as early stage, whereas expanded, hatching and hatched blastocysts were grouped as advanced stage. There was a significant reduction in the HS group of early stage blastocysts on Day 7 and of advanced stage blastocysts on Days 7 and 8. However, in Day-8 blastocysts, there was no significant difference in gene expression for any target gene. Moreover, there were no significant differences in total number of cells or apoptosis cell ratio in blastocysts. However, the ICM/TE ratio was significantly higher (P=0.021) in control (0.7) compared with HS (0.56). Sperm samples collected in August had reduced fertility compared with those obtained in March. Although fewer sperm abnormalities were present in HS, based on decreased blastocyst rates and ICM/TE ratio in embryos produced with HS semen, we inferred that molecular mechanisms for advanced blastocyst development were affected. However, those mechanisms did not involve our target genes. This work was funded by the European Union, Horizon 2020 Marie Sklodowska-Curie Action, REPBIOTECH 675526.