Cytosine methylation of sperm DNA in horse semen after cryopreservation

Aurich, Christine; Schreiner, Bettina; Ille, Natascha; Alvarenga, Marco Antônio; Scarlet, D.

doi:10.1016/j.theriogenology.2016.04.077

Cited by 50 publications

(50 citation statements)

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“…Cryopreservation may also directly affect the epigenome of the paternal DNA; recent studies have shown that cryopreservation increases the level of DNA methylation in equine sperm [10] and the expression of genes important to intracellular regulation of epigenetic status [37]. Notably, we also found significant reduction in abundance of transcripts for histones in CRYO embryos.…”

Section: Discussionsupporting

confidence: 59%

“…Exploration of differentially-expressed genes at the molecular and cellular level revealed alterations in important functions including ATP synthesis, regulation of transcription, nucleosome assembly, chromatin silencing, protein synthesis, and redox regulation. Alterations in these genes help to explain the reduced fertility observed with cryopreserved sperm attributable to increased early embryo mortality [9, 10].…”

Section: Discussionmentioning

confidence: 99%

“…Cryopreservation leads to extensive damage of sperm cell membranes and causes metabolic and functional alteration of sperm [5, 6], particularly of their mitochondria [7-9]. Cryopreservation may alter sperm DNA [10]; recently, specific cryodamage to sperm genes and transcripts have been reported [11, 12], even in samples with good sperm motility post thaw and in the absence of detectable DNA fragmentation. The sperm DNA is epigenetically programmed to regulate embryonic gene expression, and changes to this epigenome cause developmental disregulation [13].…”

Section: Introductionmentioning

confidence: 99%

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Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse

Ortiz-Rodríguez

Ortega-Ferrusola

Gil

et al. 2019

Preprint

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29 30 31 32 33 34 35 ABSTRACT 3637 Artificial insemination with cryopreserved sperm is a major assisted reproductive 38 technology in many species. In horses, as in humans, insemination with cryopreserved 39 sperm is associated with lower pregnancy rates than those for fresh sperm, however, 40 direct effects of sperm cryopreservation on the development of resulting embryos are 41 largely unexplored. The aim of this study was to investigate differences in gene 42 expression between embryos resulting from fertilization with fresh or cryopreserved 43 sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares 44 inseminated post-ovulation on successive cycles with either fresh sperm or frozen-45 thawed sperm from the same stallion, providing matched embryo pairs at each day.46 RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA 47 libraries were built and sequenced. Significant differences in transcripts per kilobase 48 million (TPM) were determined using (i) genes for which the expression difference 49 between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) 50 genes for which the fold change was  2, to avoid expression bias in selection of the 51 candidate genes. Molecular pathways were explored using the DAVID webserver, 52 followed by network analyses using STRING, with a threshold of 0.700 for positive 53 interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm 54 differed significantly from that for embryos derived from fresh sperm on all days, 55 showing significant down-regulation of genes involved in biological pathways related to 56 oxidative phosphorylation, DNA binding, DNA replication, and immune response.57 Many genes with reduced expression were orthologs of genes known to be embryonic 58 lethal in mice. This study, for the first time, provides evidence of altered transcription in 59 embryos resulting from fertilization with cryopreserved spermatozoa in any species. As 60 sperm cryopreservation is commonly used in many species, including human, the effect 61 of this intervention on expression of developmentally important genes in resulting 62 embryos warrants attention. 63 64 65 67 INTRODUCTION 68 69 Cryopreservation is a common procedure in assisted reproductive technology, in both 70 humans and the animal breeding industry [1-3]. Cryopreserved sperm are routinely used 71 for artificial insemination (AI), in vitro fertilization (IVF) and intracytoplasmic sperm 72 injection (ICSI). However, it is clear that sperm cryopreservation methods are currently 73 sub-optimal, as pregnancy rates with cryopreserved sperm are lower than those with 74 fresh sperm in humans and horses [4], among other species. Cryopreservation leads to 75 extensive damage of sperm cell membranes and causes metabolic and functional 76 alteration of sperm [5, 6], particularly of their mitochondria [7-9]. Cryopreservation 77 may alter sperm DNA [10]; recently, specific cryodamage to sperm genes and 78 transcripts have been reported [11,...

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Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse

Ortiz-Rodríguez

Ortega-Ferrusola

Gil

et al. 2019

Preprint

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“…In addition, the effects of some commonly used techniques has still not been evaluated. Very recently, for example, Aurich, Schreiner, Ille, Alvarenga, and Scarlet () showed that semen freezing increased DNA methylation in horses, whatever the cryopreservative solution used. Finally, new techniques are constantly being developed such as in vitro maturation of human oocytes, ooctyte freezing, cytoplasmic or pronuclear transfer and these need to be evaluated.…”

Section: Resultsmentioning

confidence: 99%

Long term effects of ART: What do animals tell us?

Duranthon

Chavatte‐Palmer

2018

Molecular Reproduction Devel

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Early stages of mammalian embryonic development are now known to be very sensitive to their microenvironment, with long term effects on fetal, postnatal, and adult health, thus extending to these early stages the concept of Developmental Origin of Health and Disease (DoHaD). In this scientific context, and with 3% of births in developed countries, safety of Assisted Reproductive Techniques procedures becomes a matter of concern. Besides, embryo technologies in domestic mammals, using huge number of embryos, do not seem to evidence heavy impacts on adult phenotypes. This paper first discusses what can or cannot be concluded from farm animal data, then develops long term effects of ART procedures (ovarian stimulation, in vitro fertilization and embryo culture) evidenced in model species (mainly mouse model). Recent literature demonstrates both individual and cumulative effects of each ART procedure on fetal and postnatal phenotypes. In a second part, because they are sources for further perturbations, immediate effects of ART on early embryo phenotypes at the cellular and molecular levels are described in both farm animals and model species. Mechanistic hypotheses supporting these ART induced phenotypic alterations are subsequently considered. Finally, taking into account interspecies differences in the mechanisms likely to be involved, the relevance of results obtained in animal models for human ART are discussed.

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“…Processing of semen for cryopreservation was carried out at room temperature (20°C) as described before (Aurich, Schreiner, Ille, Alvarenga, & Scarlet, 2016). The sperm pellet was partially resuspended in the remaining supernatant and split into three aliquots each diluted at a 1:1 ratio with the three cryopreservation extenders, respectively.…”

Section: Cryopreservation and Thawing Of Semenmentioning

confidence: 99%

Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post‐thaw sperm quality in stallions

Oliveira

Budik

Aurich

2017

Reprod Domestic Animals

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Although glycerol is the cryoprotectant most commonly used in stallions, it has also a considerable toxicity for equine sperm. It was the aim of this study to analyse the quality of frozen-thawed stallion semen after complete or partial replacement of glycerol in the freezing extender by alternative cryoprotectants. We hypothesized that partial or total replacement of glycerol by cryoprotectants occurring in cold-resistant frog, insect or plant species results in similar or better semen quality after freezing-thawing. As basic medium, the commercial Ghent basic extender was used and either supplemented with glucose and urea, trehalose and proline, or trehalose and betaine. Based on a series of preliminary experiments, semen was frozen in either commercial Ghent cryopreservation extender (Ghent control), Ghent glucose-urea extender or a Ghent combined extender (glucose-urea, trehalose-betaine and trehalose-proline; volume ratio of 2:1:2) in a computer-controlled rate freezer. After freezing-thawing, semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential and chromatin condensation. No differences between Ghent control and Ghent glucose-urea extender were seen, while all endpoints except DNA integrity were negatively affected in Ghent combined extender (e.g., progressive motility: Ghent 49.2 ± 3.7, Ghent glucose-urea 46.5 ± 4.6, Ghent combined 24.4 ± 2.8%; p < .001). In conclusion, glycerol concentration in a commercial freezing extender for equine spermatozoa can be successfully reduced when urea as an additive cryoprotectant is added and the glucose concentration is elevated. However, total glycerol replacement with urea, betaine, proline and trehalose was less successful.

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Cytosine methylation of sperm DNA in horse semen after cryopreservation

Cited by 50 publications

References 34 publications

Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse

Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse

Long term effects of ART: What do animals tell us?

Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post‐thaw sperm quality in stallions

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