Oocyte maturation under lipotoxic conditions induces carryover transcriptomic and functional alterations during post-hatching development of good-quality blastocysts: novel insights from a bovine embryo-transfer model

Abstract

STUDY QUESTION Does oocyte maturation under lipolytic conditions have detrimental carry-over effects on post-hatching embryo development of good-quality blastocysts after transfer? SUMMARY ANSWER Surviving, morphologically normal blastocysts derived from bovine oocytes that matured under lipotoxic conditions exhibit long-lasting cellular dysfunction at the transcriptomic and metabolic levels, which coincides with retarded post-hatching embryo development. WHAT IS KNOWN ALREADY There is increasing evidence showing that following maturation in pathophysiologically relevant lipotoxic conditions (as in obesity or metabolic syndrome), surviving blastocysts of good (transferable) morphological quality have persistent transcriptomic and epigenetic alteration even when in vitro embryo culture takes place under standard conditions. However, very little is known about subsequent development in the uterus after transfer. STUDY DESIGN, SIZE, DURATION Bovine oocytes were matured in vitro in the presence of pathophysiologically relevant, high non-esterified fatty acid (NEFA) concentrations (HIGH PA), or in basal NEFA concentrations (BASAL) as a physiological control. Eight healthy multiparous non-lactating Holstein cows were used for embryo transfers. Good-quality blastocysts (pools of eight) were transferred per cow, and cows were crossed over for treatments in the next replicate. Embryos were recovered 7 days later and assessed for post-hatching development, phenotypic features and gene expression profile. Blastocysts from solvent-free and NEFA-free maturation (CONTROL) were also tested for comparison. PARTICIPANTS/MATERIALS, SETTING, METHODS Recovered Day 14 embryos were morphologically assessed and dissected into embryonic disk (ED) and extraembryonic tissue (EXT). Samples of EXT were cultured for 24 h to assess cellular metabolic activity (glucose and pyruvate consumption and lactate production) and embryos’ ability to signal for maternal recognition of pregnancy (interferon-τ secretion; IFN-τ). ED and EXT samples were subjected to RNA sequencing to evaluate the genome-wide transcriptome patterns. MAIN RESULTS AND THE ROLE OF CHANCE The embryo recovery rate at Day 14 p.i. was not significantly different among treatment groups (P > 0.1). However, higher proportions of HIGH PA embryos were retarded in growth (in spherical stage) compared to the more elongated tubular stage embryos in the BASAL group (P < 0.05). Focusing on the normally developed tubular embryos in both groups, HIGH PA exposure resulted in altered cellular metabolism and altered transcriptome profile particularly in pathways related to redox-regulating mechanisms, apoptosis, cellular growth, interaction and differentiation, energy metabolism and epigenetic mechanisms, compared to BASAL embryos. Maturation under BASAL conditions did not have any significant effects on post-hatching development and cellular functions compared to CONTROL. LARGE-SCALE DATA The datasets of RNA sequencing analysis are available in the NCBI’s Gene Expression Omnibus (GEO) repository, series accession number GSE127889 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127889). Datasets of differentially expressed genes and their gene ontology functions are available in the Mendeley datasets at http://dx.doi.org/10.17632/my2z7dvk9j.2. LIMITATIONS, REASONS FOR CAUTION The bovine model was used here to allow non-invasive embryo transfer and post-hatching recovery on Day 14. There are physiological differences in some characteristics of post-hatching embryo development between human and cows, such as embryo elongation and trophoblastic invasion. However, the main carry-over effects of oocyte maturation under lipolytic conditions described here are evident at the cellular level and therefore may also occur during post-hatching development in other species including humans. In addition, post-hatching development was studied here under a healthy uterine environment to focus on carry-over effects originating from the oocyte, whereas additional detrimental effects may be induced by maternal metabolic disorders due to adverse changes in the uterine microenvironment. RNA sequencing results were not verified by qPCR, and no solvent control was included. WIDER IMPLICATIONS OF THE FINDINGS Our observations may increase the awareness of the importance of maternal metabolic stress at the level of the preovulatory oocyte in relation to carry-over effects that may persist in the transferrable embryos. It should further stimulate new research about preventive and protective strategies to optimize maternal metabolic health around conception to maximize embryo viability and thus fertility outcome. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Flemish Research Fund (FWO grant 11L8716N and FWO project 42/FAO10300/6541). The authors declare there are no conflicts of interest.

The Metabolic Signature of In Vitro Produced Bovine Embryos Helps Predict Pregnancy and Birth after Embryo Transfer

“…These events are conceptually similar to the dynamic reprogramming during embryonic development, by which environmental stimuli (including assisted reproductive technologies) lead to acquisition of a stable, modified genotype later in life [ 59 ] and, in this case, loss of pregnancy competence. NEFAs are mainly responsible for low embryonic fitness [ 24 , 60 , 61 , 62 , 63 ]. Our results confirm findings with V/W embryos [ 24 ] in which fully viable embryos were those with less active lipid catabolism, as shown by reduced FA contents in their CM.…”

Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates

“…Embryo transfer of in vitro produced (IVP) embryos often results in lower pregnancy rates compared to their in vivo counterparts, as it will be discussed below, and failures in epiblast development seem to be the main responsible for such developmental arrest. IVP-derived bovine conceptuses have been reported to exhibit smaller EDs than their in vivo counterparts ( 84 ), and multiple studies have reported a remarkably high percentage (23–65%) of IVP-derived conceptuses lacking EDs ( 4 , 85 – 90 ), as reviewed by Ealy et al ( 91 ). Impaired ED development has also been observed in ovine and bovine embryos produced by somatic cell nuclear transfer (SCNT), a technology that induces pleiotropic effects over different lineages ( 92 , 93 ).…”

“…During the next days, the ED will acquire an oval shape and a higher density at the posterior edge, associated with the ingression of the first cells into the primitive streak and the beginning of gastrulation. Some cells at the posterior part of the epiblast start to express the mesoderm marker BRACHYURY (T) and to downregulate SOX2 before the primitive streak is morphologically visible (59,(69)(70)(71)(72)). These T-positive cells will be the first cells to egress into the space between the epiblast and the hypoblast and will form the mesoderm, which quickly migrates to cover the whole embryonic disc.…”

“…This pioneer system paves the way for future research focused on improving the conditions of in vitro embryo production or associated techniques such as embryo freezing or vitrification, as it allows direct embryo quality assessment without the need of experimental animals. The potential roles on lineages development of specific metabolites, hormones, or growth factors whose levels are altered in detrimental conditions for embryo survival, such as negative energy balance in post-partum dairy cows ( 90 ), can also be analyzed in this system.…”

Comparing in vitro maturation rates in buffalo and cattle oocytes and evaluating the effect of cAMP modulators on maturation and subsequent developmental competence

“…The lack of synchronization between nuclear and cytoplasmic maturation, as well as insufficient cytoplasmic maturation, results in oocytes matured in vitro having lower developmental competence than those matured in vivo [3,4,6,7]. Despite extensive research into factors affecting oocyte maturation in mice and other animals, the molecular mechanism governing oocyte maturation in buffalo remains unknown [8][9][10][11].…”