Efficiency of embryo complementation and pluripotency maintenance following multiple passaging of in vitro-derived bovine embryos.

Context Current methods to obtain bovine embryos of high genetic merit include approaches that require skilled techniques for low-efficiency cloning strategies. Aims The overall goal herein was to identify the efficacy of alternative methods for producing multiple embryos through blastomere complementation while determining maintenance of cell pluripotency. Methods Bovine oocytes were fertilised in vitro to produce 4-cell embryos from which blastomeres were isolated and cultured as 2-cell aggregates using a well-of-the-well system. Aggregates were returned to incubation up to 7days (Passage 1). A second passage of complement embryos was achieved by splitting 4-cell Passage 1 embryos. Passaged embryos reaching the blastocyst stage were characterised for cell number and cell lineage specification in replicate with non-reconstructed zona-intact embryos. Key results Passage 1 and 2 embryo complements yielded 29% and 25% blastocyst development, respectively. Passage 1 embryos formed blastocysts, but with a reduction in expression of SOX2 and decreased size compared to non-reconstructed zona-intact embryos. Passage 2 embryos had a complete lack of SOX2 expression and a reduction in transcript abundance of SOX2 and SOX17, suggesting loss of pluripotency markers that primarily affected inner cell mass (ICM) and hypoblast formation. Conclusions In vitro fertilised bovine embryos can be reconstructed with multiple passaging to generate genetically identical embryos. Increased passaging drives trophectoderm cell lineage specification while compromising ICM formation. Implications These results may provide an alternative strategy for producing genetically identical bovine embryos through blastomere complementation with applications towards the development of trophoblast and placental models of early development.

PPAR-gamma influences developmental competence and trophectoderm lineage specification in bovine embryos.

In brief: Peroxisome proliferator-activated receptor gamma (PPARG) is a critical regulator of placental function, but earlier roles in preimplantation embryo development and embryonic origins of placental formation have not been established. Results herein demonstrate that PPARG responds to pharmacologic stimulation in the bovine preimplantation embryo and influences blastocyst development, cell lineage specification, and transcripts important for placental function. Abstract: Peroxisome proliferator-activated receptor gamma (PPARG) is a key regulator of metabolism with conserved roles that are indispensable for placental function, suggesting previously unidentified and important roles in preimplantation embryo development. Herein, we report the functional characterization of bovine PPARG to reveal expression beginning on D6 of development with nuclear and ubiquitous patterns. Day 6 PPARG+ embryos have fewer total cells and a lower proportion of trophectoderm cells compared to PPARG- embryos (P < 0.05). Coculture with a PPARG agonist, rosiglitazone (Ros), or antagonist GW9662 (GW), decreases blastocyst development (P < 0.01). Day 7.5 (D7.5) developmentally delayed embryos exposed to Ros express lower transcript abundance of key genes important for placental development and cell lineage formation (CDX2, RXRB, SP1, TFAP2C, SIRT1, and PTEN). In contrast, Ros does not alter transcript abundance in D7.5 blastocysts, but GW treatment lowers RXRA, RXRB, SP1, and NFKB1 expression. Knockout of embryonic PPARG does not alter blastocyst formation and hatching ability but decreases total cell number in D7.5 blastocysts. The decreased embryo development response and affected pathways following targeted pharmacological perturbation vs embryonic knockout of PPARG suggest roles of both maternal and embryonic origins. These data reveal regulatory contributions of PPARG in preimplantation embryo development, cell lineage formation, and regulation of transcripts associated with placental function.

Environment to embryo: intersections of contaminant exposure and preimplantation embryo development in agricultural animals†.

Environmental impacts on reproductive function are well documented in humans, yet little information is known about the effects on large animals. The interface of environment and reproduction has evolved prudently with a concerted effort to ensure global food sustainability tightly integrated with the application of technological advances in agriculture production that include nutrient and resource management. Exposure to environmental toxicants through chemical pesticide application and industry practices has coincided with a decline in cattle and human fertility. The increased adoption of agriculture animals for human biomedical models further emphasizes the importance of understanding the consequences of livestock exposure to environmentally and physiologically relevant levels of contaminants to preimplantation embryo development. In addition, increased awareness of paternal contributions to the early embryo that include both genetic and nongenetic factors supports the need to define environmental interactions from gamete to genome. Herein we summarize current knowledge of common environmental contaminants on reproductive function including direct and indirect effects on embryo development success in livestock. Information obtained from a diverse number of species including humans is presented to illustrate gaps in knowledge within livestock directly pertaining to agriculture success, sustainability, clinical practice, and biomedical research.