Genes relevant for early embryo development in horses are down-regulated when frozen semen is used, researchers have found.
Down-regulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, which are coded by the genes.
Fernando Peña and his colleagues, writing in the open-access journal PLOS ONE, acknowledged the preliminary and descriptive nature of their report.
“But our data provide strong evidence that cryopreservation of sperm exerts a profound impact on the transcriptome (the set of all RNA molecules) of early embryos.
“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,” they said.
The researchers said artificial insemination with cryopreserved spermatozoa – more commonly called frozen semen – is a major assisted reproductive technology in many species.
They said the contribution of sperm to embryo development has evolved from the concept that the only role of sperm at fertilization is to introduce the male genome into the egg.
Sperm, they said, carry a myriad of small noncoding RNAs with potential roles in early embryo development.
In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, suggesting that preservation methods are currently sub-optimal. It leads to damage to sperm cell membranes and causes metabolic and functional alteration of sperm, particularly of their mitochondria.
However, the direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored, they noted.
In a study based at the University of Extremadura in Spain, scientists set out 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 on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, effectively providing matched embryo pairs at each day.
RNA was isolated from two matched pairs (4 embryos) for each day for analysis.
The transcriptional profile – gene expression at the RNA level – 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 (an important mechanism by which the activity of proteins can be altered after they are formed), DNA binding, DNA replication, and immune response.
The authors say their work provides the first evidence of altered transcription in embryos resulting from fertilization with frozen semen in any species.
Discussing their findings, the study team said the evidence showed that procedures performed during the handling of sperm, such as freezing and thawing, have a significant impact on critical aspects of the early embryo transcriptome.
“While the mechanisms behind the effects reported here are as yet unclear, a major factor may be the well-documented oxidative damage that the genome and epigenome experiences during cryopreservation and thawing.” Cryopreservation is a major cause of oxidative stress and lipid peroxidation (degradation) in stallion sperm, they said.
The full study team comprised Peña, José Ortiz-Rodriguez, Cristina Ortega-Ferrusola, María Gil, Francisco Martín-Cano, Gemma Gaitskell-Phillips, Alberto Álvarez-Barrientos, and Ángel Román (all with the University of Extremadura), and Heriberto Rodríguez-Martínez (Linköping University in Sweden), and Katrin Hinrichs (Texas A&M University).
Ortiz-Rodriguez JM, Ortega-Ferrusola C, Gil MC, Martín-Cano FE, Gaitskell-Phillips G, Rodríguez-Martínez H, et al. (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