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.

Impact of oxidative stress on male and female germ cells: implications for fertility

“…In addition, sublethal levels of oxidative stress are known to impact the integrity of sperm DNA thereby influencing the potential development of the embryo and the health and wellbeing of the offspring (Aitken et al 1998a, Aitken & Curry 2011. For example, the altered patterns of gene expression observed in equine embryos as a consequence of insemination with cryostored spermatozoa maybe a case in point, because cryostorage is known to be associated with the induction of oxidative DNA damage (Thomson et al 2009, Ortiz-Rodriguez et al 2019.…”

Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality

“…In horses, sperm cryopreservation increased global DNA methylation [77], whereas in boar sperm, freezing-thawing decreased the relative abundance of mRNAs as well as the protein levels of some genes associated with DNA methylation (DNMT3A, DNMT3B), histone modifications (JHDM2A, KAT8) and genomic imprinting (IGF2) [78]. Recently, a comparative transcriptome analysis of embryos obtained with fresh and cryopreserved stallion sperm revealed that several sperm mRNA transcripts with a relevant function in early embryo development were downregulated after cryopreservation [79]. However, there is a lack of similar studies in ruminant species.…”

Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa

“…Animal studies indicate that fecundation with spermatozoa experiencing oxidative stress may cause embryonic death [203], an effect that has been linked to oxidative damage in the spermatozoa [204]. Recent research from our laboratory has compared the effect of cryopreservation on the transcriptome of early equine embryos [205]. Using the same ejaculate, half processed as fresh sperm and the other half frozen and thawed, we obtained embryos from the same mare and stallion after artificial insemination with the aliquot of fresh sperm and, in the mare’s next cycle using the frozen thawed semen aliquot.…”

“…Significant downregulation of genes involved in biological pathways related to the gene ontology (GO) terms oxidative phosphorylation, DNA binding, DNA replication, and immune response . Interestingly, many genes with reduced expression were orthologs of genes in which knockouts are embryonic lethal in mice [205]. While the exact mechanism behind these changes remains to be elucidated, redox deregulation and oxidative stress in the spermatozoa seem to be an important factor.…”

“…Even though further investigation is needed in this field, oxidative damage to sperm proteins with important functions during early embryo development may occur. Further supporting this hypothesis is the fact that biological processes such as DNA binding and replication, and Histone Acetylation were downregulated in embryos obtained with cryopreserved spermatozoa [205], and many of the proteins mentioned above have roles in these processes [201].…”