Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

PURPOSE: Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. METHODS: We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2 h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. RESULTS: In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. CONCLUSIONS: Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

Oocytes, embryos and pluripotent stem cells from a biomedical perspective.

The veterinary and animal science professions are rapidly developing and their inherent and historical connection to agriculture is challenged by more biomedical and medical directions of research. While some consider this development as a risk of losing identity, it may also be seen as an opportunity for developing further and more sophisticated competences that may ultimately feed back to veterinary and animal science in a synergistic way. The present review describes how agriculture-related studies on bovine in vitro embryo production through studies of putative bovine and porcine embryonic stem cells led the way to more sophisticated studies of human induced pluripotent stem cells (iPSCs) using e.g. gene editing for modeling of neurodegeneration in man. However, instead of being a blind diversion from veterinary and animal science into medicine, these advanced studies of human iPSC-derived neurons build a set of competences that allowed us, in a more competent way, to focus on novel aspects of more veterinary and agricultural relevance in the form of porcine and canine iPSCs. These types of animal stem cells are of biomedical importance for modeling of iPSC-based therapy in man, but in particular the canine iPSCs are also important for understanding and modeling canine diseases, as e.g. canine cognitive dysfunction, for the benefit and therapy of dogs.

Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cell transcriptomics.

The Ovum Pick Up-In vitro Production (OPU-IVP) of embryos is an advanced reproductive technology used in cattle production but the complex biological mechanisms behind IVP outcomes are not fully understood. In this study we sequenced RNA of granulosa cells collected from Holstein cows at oocyte aspiration prior to IVP, to identify candidate genes and biological mechanisms for favourable IVP-related traits in donor cows where IVP was performed separately for each animal. We identified 56 genes significantly associated with IVP scores (BL rate, kinetic and morphology). Among these, BEX2, HEY2, RGN, TNFAIP6 and TXNDC11 were negatively associated while Mx1 and STC1 were positively associated with all IVP scores. Functional analysis highlighted a wide range of biological mechanisms including apoptosis, cell development and proliferation and four key upstream regulators (COX2, IL1, PRL, TRIM24) involved in these mechanisms. We found a range of evidence that good IVP outcome is positively correlated with early follicular atresia. Furthermore we showed that high genetic index bulls can be used in breeding without reducing the IVP performances. These findings can contribute to the development of biomarkers from follicular fluid content and to improving Genomic Selection (GS) methods that utilize functional information in cattle breeding, allowing a widespread large scale application of GS-IVP.

Proteomic analysis of bovine blastocoel fluid and blastocyst cells.

The understanding of the early mammalian development is a prerequisite for the advancement of in vitro fertilization and improvement of derivation and culturing of embryonic stem cells. While, whole genome transcriptomic analysis on bovine blastocysts has identified genes active in early development, little information is available about the protein complement of early embryos. Modern, sensitive proteomic technology (nano HPLC tandem mass spectrometry) allowed us to describe the proteome of the scarce blastocoel fluid and cell material of expanded bovine blastocysts isolated by micromanipulation. From two independent replicates, 23 proteins were identified in the blastocoel fluid while 803 proteins were identified in the remaining cell material. The proteins were grouped into categories according to their gene ontology (GO) terms by which proteins involved in cell differentiation, cell proliferation, development, and reproduction could be derived. Proteins classified in these categories could be candidates for further functional studies to understand pluripotency and early mammalian development.

Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos.

The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early and slightly later stage cell differentiation in the developing bovine embryo. In vitro-produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a considerable contribution to the current knowledge of bovine preimplantation development.