Spermatozoa telomeres determine telomere length in early embryos and offspring

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth. © 2016 Society for Reproduction and Fertility

Cultured bovine embryo biopsy conserves methylation marks from original embryo

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. Thiswork analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ~4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cellmass were separated.We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation ismore similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved

Embryo culture in presence of oviductal fluid induces DNA methylation changes in bovine blastocysts

During the transit through the oviduct, the early embryo initiates an extensive DNA methylation reprogramming of its genome. Given that these epigenetic modifications are susceptible to environmental factors, components present in the oviductal milieu could affect the DNA methylation marks of the developing embryo. The aim of this study was to examine if culture of bovine embryos with oviductal fluid (OF) can induce DNA methylation changes at specific genomic regions in the resulting blastocysts. In vitro produced zygotes were cultured in medium with 3 mg/mL bovine serum albumin (BSA) or 1.25% OF added at the one- to 16-cell stage (OF1-16), one- to 8-cell stage (OF1-8) or 8- to 16-cell stage (OF8-16), and then were cultured until Day 8 in medium with 3 mg/mL BSA. Genomic regions in four developmentally important genes (MTERF2, ABCA7, OLFM1, GMDS) and within LINE-1 retrotransposons were selected for methylation analysis by bisulfite sequencing on Day 7-8 blastocysts. Blastocysts derived from OF1-16 group showed lower CpG methylation levels in MTERF2 and ABCA7 compared with the BSA group. However, CpG sites within MTERF2, ABCA7 and OLFM1 showed higher methylation levels in groups OF1-8 and OF8-16 than in OF1-16. For LINE-1 elements, higher CpG methylation levels were observed in blastocysts from the OF1-16 group than in the other experimental groups. In correlation with the methylation changes observed, mRNA expression level of MTERF2 was increased, while LINE-1 showed a decreased expression in blastocysts from OF1-16 group. Our results suggest that embryos show transient sensitivity to OF at early stages, which is reflected by specific methylation changes at the blastocyst stage. © 2017 Society for Reproduction and Fertility

ZP4 confers structural properties to the zona pellucida essential for embryo development

18 Pág.Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.This work was supported by Grants AGL2014-58739-R, RYC-2012-10193, AGL2017-84908R, AGL2016-71890-REDT, PGC2018-094781-B-100, AGL2015-70159-P and AGL2015-65572-C2-1-R from the Spanish Ministries of Economy and Competitiveness and Science, Innovation and University, and 757886-ELONGAN from the European Research Council. ILT and NFB are supported by FPI fellowships from the Spanish Ministry of Economy and Competitiveness. AQF by Marie Curie Action FP7/2007-2013 grant 600391 from EU, and LGB by a FPU fellowship from the Spanish Ministry of Education, Culture and sportsPeer reviewe

Potential health risks associated to ICSI Insights from animal models and strategies for a safe procedure

Artificial reproductive techniques are currently responsible for 1.7-4% of the births in developed countries and intracytoplasmatic sperm injection (ICSI) is the most commonly used, accounting for 70-80% of the cycles performed. Despite being an invaluable tool for infertile couples, the technique bypasses several biological barriers that naturally select the gametes to achieve an optimal embryonic and fetal development. In this perspective, ICSI has been associated with an increased risk for diverse health problems, ranging from premature births and diverse metabolic disorders in the offspring to more severe complications such as abortions, congenital malformations, and imprinting disorders. In this review, we discuss the possible implications of the technique per se on these adverse outcomes and highlight the importance of several experiments using mammalian models to truthfully test these implications and to uncover the molecular base that origins these health problems. We also dissect the specific hazards associated to ICSI and describe some strategies that have been developed to mimic the gamete selection occurring in natural conception in order to improve the safety of the procedure. © 2014 Sánchez-Calabuig, López-Cardona, Fernández-González, Ramos-Ibeas, FonsecaBalvís, Laguna-Barraza, Pericuesta, Gutiérrez-Adánand Bermejo-álvarez