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In this essay, we propose that embryos express a metabolic phenotype necessarily different from that of differentiated somatic cells and more like that of rapidly proliferating cancer cells. This metabolic adaptation, known as the Warburg Effect, supports rapid cell proliferation. One of the hallmarks of the Warburg Effect is that pyruvate is directed away from the tri-carboxylic acid cycle and metabolized to lactate, resulting in a buildup of glycolytic intermediates. Although this is a comparatively inefficient way to generate ATP, this adaptation allows the cell to meet other critical metabolic requirements, including biomass production and redox regulation. Thus, utilization of WE gives proliferating cells a selective growth advantage. This model represents a completely new understanding of embryo metabolism in the context of a broad, interconnected network of metabolic mechanisms that influence viability, versus the current dogma of carbohydrate metabolism via oxidative phosphorylation. A more complete understanding of embryo metabolism is critical to better support embryo viability in vitro, and to avoid forcing embryos to adapt to suboptimal culture conditions at a significant cost to future growth and development.

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The objective of these experiments was to evaluate the importance of fatty acid beta-oxidation (FAO) in the cumulus oocyte complex (COC) during in vitro maturation (IVM) to oocyte nuclear maturation and gene expression in both the oocyte and cumulus cells in three species with differing amounts of oocyte intracellular lipids (mouse, low; bovine, moderate; porcine, high). We inhibited FAO using etomoxir at 0, 10, 25, 100, or 250 lM. Completion of oocyte nuclear maturation was inhibited after COC exposure to 250 lM etomoxir in mouse oocytes, 100 lM etomoxir in bovine oocytes, and as little as 10 lM etomoxir in porcine oocytes (P , 0.05). When FAO was inhibited in mouse and porcine COCs resulting in inhibition of meiosis, the abundance of FAO, glycolytic, and oxidative stress gene transcripts were decreased in oocytes and cumulus cells (P , 0.05), although to a much greater extent in the pig. In bovine oocytes and cumulus cells, FAO gene transcripts were increased and glycolytic gene expression altered following meiotic inhibition due to etomoxir. Etomoxir, at doses that did not inhibit nuclear maturation in bovine and murine COCs, increased glucose consumption (P , 0.05), suggesting glucose metabolism is increased to meet the metabolic demands of the COCs when fatty acid metabolism is compromised. Our data demonstrates that FAO is essential to oocyte nuclear maturation in all three species. Sensitivity of nuclear maturation to FAO inhibition reflects the amount of lipid present in the ooplasm and may suggest a relative reliance on this metabolic pathway.etomoxir, fatty acid oxidation, meiosis, oocyte maturation

Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.

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Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.

Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.