Molecular fingerprint of female bovine embryos produced in vitro with high competence to establish and maintain pregnancy†

Abstract

<jats:title>Abstract</jats:title> <jats:p>The objective was to identify the transcriptomic profile of in vitro-derived embryos with high competence to establish and maintain gestation. Embryos produced with X-sorted sperm were cultured from day 5 to day 7 in serum-free medium containing 10 ng/ml recombinant bovine colony-stimulating factor 2 (CSF2) or vehicle. The CSF2 was administered because this molecule can increase blastocyst competence for survival after embryo transfer. Blastocysts were harvested on day 7 of culture and manually bisected. One demi-embryo from a single blastocyst was transferred into a synchronized recipient and the other half was used for RNA-seq analysis. Using P &amp;lt; 0.01 and a fold change &amp;gt;2-fold or &amp;lt;0.5 fold as cutoffs, there were 617 differentially expressed genes (DEG) between embryos that survived to day 30 of gestation vs those that did not, 470 DEG between embryos that survived to day 60 and those that did not, 432 DEG between embryos that maintained pregnancy from day 30 to day 60 vs those where pregnancy failed after day 30, and 635 DEG regulated by CSF2. Pathways and ontologies in which DEG were overrepresented included many related to cellular responses to stress and cell survival. It was concluded that gene expression in the blastocyst is different between embryos that are competent to establish and maintain pregnancy vs those that are not. The relationship between expression of genes related to cell stress and subsequent embryonic survival probably reflects cellular perturbations caused by embryonic development taking place in the artificial environment associated with cell culture.</jats:p>

The incompletely fulfilled promise of embryo transfer in cattle—why aren’t pregnancy rates greater and what can we do about it?

“…The approach has been to identify genes whose expression is associated with embryo survival in bisected embryos, where one demi-embryo is transferred into a recipient and the other is used for analysis of the transcriptome. This type of analysis has been performed for both MOET embryos ( Salilew-Wondim et al 2010 , Ghanem et al, 2011 ; Zolini et al, 2020a ) and IVP embryos ( El-Sayed et al, 2006 ; Zolini et al, 2020b ). In each experiment, a panel of genes was identified whose expression was associated with embryo survival after transfer.…”

“…The embryo production system is also likely to influence genes associated with embryo survival. For female Holstein embryos, many genes associated with survival of MOET embryos were related to low oxygen consumption and possible differentiation of the inner cell mass to epiblast ( Zolini et al, 2020a ), whereas many genes associated with survival of IVP embryos were related to cell stress and cell survival ( Zolini et al, 2020b ).…”

Metabolites Secreted by Bovine Embryos In Vitro Predict Pregnancies That the Recipient Plasma Metabolome Cannot, and Vice Versa

“…Thus, collection of embryonic cells by biopsy allows the investigation of chromosomal stability [ 9 ] and the expression of genes regulated in single embryos with higher competence to develop [ 10 , 11 ]. Biopsy is, nevertheless, invasive, and the damage done to the embryo may reduce pregnancy rates, potentially leading to misidentification of embryos with actual pregnancy potential [ 10 , 12 ].…”

Determinants of survival of the bovine blastocyst to cryopreservation stress: treatment with colony stimulating factor 2 during the morula-to-blastocyst transition and embryo sex

“…Treatment with CSF2 reduced the induction of apoptosis in Day 6 bovine embryos caused by heat shock ( Loureiro et al 2011 ). Moreover, a large fraction of the 635 genes whose expression was regulated by CSF2 in female blastocysts are involved in processes related to cell stress ( Zolini et al 2020 ). In the mouse, too, CSF2 treatment can reduce expression of genes involved in apoptosis and the heat shock protein response ( Chin et al 2009 ).…”

“…Damage to the embryo is multifactorial and includes ultrastructural damage from ice crystals (in slow freezing), cytotoxic effects of cryoprotectants, damage to lipid membranes caused by phase transition, lipid peroxidation, and induction of apoptosis after thawing ( Ferré et al 2020 ). It was hypothesized that cryosurvival of embryos treated with CSF2 from day 5 to 7 of development, a time when the embryo is progressing from the morula to blastocyst stage ( Hansen and Tríbulo 2019 ) and when CSF2 can affect development, gene expression, and sensitivity to apoptosis ( Moraes and Hansen 1997 ; Loureiro et al 2009 ; Dobbs et al 2013 ; Siqueira and Hansen 2016 ; Loureiro et al 2011 ; Zolini et al 2020 ), would increase the proportion of embryos that survived vitrification. The concentration of CSF2 used, 10 ng/mL, used was shown to protect embryos from heat shock ( Loureiro et al 2011 ) and induce expression of genes related to cell stress ( Zolini et al 2020 ).…”