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Home / Equine Research Bank / PLoS One / Transcriptome analysis reveals that fertilization with cryop...
PloS one2019; 14(6); e0213420; doi: 10.1371/journal.pone.0213420

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

Abstract: Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ≥ 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.
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Publication Date: 2019-06-25 PubMed ID: 31237882PubMed Central: PMC6592594DOI: 10.1371/journal.pone.0213420Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research demonstrates that using frozen sperm for fertilization in horses results in significant changes in gene expression in the resulting embryos, which may explain lower pregnancy rates compared to fresh sperm. The genes affected play a crucial role in early embryonic development.

Objective of the Research

  • In this study, scientists aimed to examine the differences in gene expression between embryos originating from the fertilization of fresh and cryopreserved (frozen and then thawed) sperm. This research was motivated by the observation that the use of frozen sperm yields lower pregnancy rates than that of fresh sperm.

Methodology

  • The study worked with horse embryos obtained at different stages (8, 10, or 12 days) after ovulation, following insemination with either fresh or frozen-thawed sperm from the same stallion. This allowed for direct comparison, eliminating any genetic variation between the two groups.
  • RNA was isolated from embryos at each stage for both types of insemination (fresh and frozen-thawed sperm). This RNA was then used to build cDNA libraries, which were sequenced to determine gene expression.
  • Differences in gene expression were calculated using specific statistical measures, with special emphasis on genes demonstrating a high degree of variance in expression or a large number of transcripts produced.

Results and Findings

  • The results showed that the transcriptional profile of embryos fertilized with frozen-thawed sperm differed significantly across all days from those fertilized with fresh sperm. Gene downregulation, or decreased expression, was observed in those embryos fertilized with frozen-thawed sperm.
  • The downregulated genes are involved in essential biological processes relevant to embryo development, including oxidative phosphorylation, DNA binding, DNA replication, and immune response. Several of these genes with reduced expression have been identified as embryonic lethal when inactivated in mice, indicating their importance in early development.

Implications of the Study

  • The results of this study provide the first evidence of altered gene expression in embryos fertilized with cryopreserved sperm, demonstrating a potential genetic mechanism for the lower pregnancy rates observed with frozen sperm use.
  • This topic holds relevancy beyond equine reproduction—it has implications for many other species, including humans, where sperm cryopreservation is routinely used. The findings highlight the need to further investigate the effects of this technology on gene expression during early embryonic development.

Cite This Article

APA
Ortiz-Rodriguez JM, Ortega-Ferrusola C, Gil MC, Martín-Cano FE, Gaitskell-Phillips G, Rodríguez-Martínez H, Hinrichs K, Álvarez-Barrientos A, Román Á, Peña FJ. (2019). Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse. PLoS One, 14(6), e0213420. https://doi.org/10.1371/journal.pone.0213420

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 14
Issue: 6
Pages: e0213420
PII: e0213420

Researcher Affiliations

Ortiz-Rodriguez, José M
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Ortega-Ferrusola, Cristina
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Gil, María C
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Martín-Cano, Francisco E
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Gaitskell-Phillips, Gemma
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Rodríguez-Martínez, Heriberto
  • Department of Clinical and Experimental Medicine, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden.
Hinrichs, Katrin
  • Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas.
Álvarez-Barrientos, Alberto
  • STAB, University of Extremadura, Badajoz, Spain.
Román, Ángel
  • Department of Biochemistry and Molecular Biology, University of Extremadura, Badajoz, Spain.
Peña, Fernando J
  • Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.

MeSH Terms

  • Animals
  • Cryopreservation / veterinary
  • Down-Regulation
  • Embryo, Mammalian
  • Embryonic Development / genetics
  • Female
  • Gene Expression Profiling
  • Horses
  • Insemination, Artificial / methods
  • Insemination, Artificial / veterinary
  • Male
  • Semen Preservation / adverse effects
  • Semen Preservation / veterinary

Conflict of Interest Statement

The authors have declared that no competing interests exist.

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