Expression profile of developmental competence gene markers in comparison with prostaglandin F2α synthesis and action in the early- and late-cleaved pre-implantation bovine embryos.

The kinetics of early cleavage stages can affect embryo quality. The bovine model of early- and late-cleaved embryos has been described in the literature and is deemed a useful tool in the field of oocyte developmental competence studies. The expression of genes demonstrating developmental potential differs between early- and late-cleaved embryos. Previously, we demonstrated that prostaglandin F2α synthase (PGFS) and prostaglandin F2α receptor (PTGFR) expression depend on the developmental stage and embryo quality. In the present study, we used the same model to determine the mRNA expression profile of developmentally important genes (IGF1R, IGF2R, PLAC8, OCT4, SOX2) in early, expanded and hatched blastocysts obtained from the early- and late-cleaved group of embryos, as well as to correlate the transcription levels of these embryonic gene markers with the transcription levels of PGFS and PTGFR. The mRNA expression of PGFS, PTGFR and factors described as gene markers of embryonic implantation ability and developmental competence genes was determined by real-time PCR. The obtained results were analysed using statistical software GraphPad prism 6.05. During the course of our analyses, we observed that the transcript abundance of most analysed genes tends to be higher in the late-rather than in the early cleaved group of embryos, as well as in B and/or C grade embryos rather than in A grade embryos. On the other hand, for the early cleaved group of blastocysts with cavity, we detected higher PLAC8 mRNA expression for grade A embryos compared with grade C embryos. It suggests that the mRNA expression level of genes depends on the quality of embryos but differs according to various factors including the method of production or culture method. Moreover, numerous correlations between analysed gene markers and PGF2α synthase and PGF2α receptor suggest that PGF2α plays a role in the crucial steps of bovine embryo development.

mRNA Expression and Role of PPARγ and PPARδ in Bovine Preimplantation Embryos Depending on the Quality and Developmental Stage.

Peroxisome proliferator-activated receptors (PPARs), a nuclear receptors for prostacyclin (PGI2) have been recognized as being essential for early embryo development. The objectives of the present study were to determine if the bovine early- and late-cleaved embryos in different stages of early development express PPARγ and PPARδ. Since embryo developmental competence depends on numerous biological factors, we evaluated if the expression of PPARγ and PPARδ correlate with selected embryo quality markers (SOX2, OCT4, PLAC8, IGF1R) in the in vitro produced embryos at different stages of their development. Developmental rates and embryo quality for early- and late-cleaved embryos were provided according to International Embryo Transfer Society (IETS; developmental stages: 2-, 4-, 16-cell embryo, morula, blastocyst (1-early, 2-developing, 3-expanded, 4-hatched); quality stages: A-high quality, B-moderate quality, C-low quality). We found that bovine embryos expressed mRNA of PPARδ and PPARγ at all stages of early development, independently of their quality. In addition, the expression of PPARδ and PPARγ correlated with the expression of quality markers in bovine blastocysts. Positive correlations were stronger and more frequent in the group of early-cleaved embryos, whereas the negative correlations were typical for the group of late-cleaved embryos. Obtained results and available literature reports may indicate the participation of PGI2, via PPARδ and PPARγ, in the processes related to the early embryo development, through the participation of this factor in the modulation of blastocyst hatching, implantation, and post-implantation development.

Iloprost affects in vitro maturation and developmental competence of bovine oocytes.

Prostacyclin (PGI2) is synthesised in oviductal fluid and enhance the embryo development during the preimplantation period. The objective of the present study was to determine the effect of an analogue of prostacyclin (iloprost) on the in vitro maturation (IVM) and the developmental competence of bovine oocytes. Cumulus oocyte complexes (COCs) were cultured in maturation medium with iloprost (0.5 µM) for 24 h. We found that iloprost assisted maturation rates and cumulus cell expansion of bovine oocytes, and it increased the mRNA expression of genes related to cumulus expansion: ADAM17, AREG, and TNFAIP6 and cathepsin genes (CTSK and CTSS). Moreover, iloprost reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2). COCs treatment with iloprost during IVM also reduced intracellular reactive oxygen species (ROS) levels, while glutathione (GSH) levels and the mRNA expression of antioxidant genes CAT and GPx4 were markedly increased. We also showed that an analogue of PGI2 influenced the mitochondrial status via distribution rates of mitochondria and mitochondrial membrane potential in oocytes. Although, iloprost-enhanced maturation had no direct effect on number of embryos cleaved, it increased blastocyst rates of bovine embryos as well as proportion of expanded blastocysts. These results indicate that the supplementation of maturation medium with iloprost is beneficial for the maturation efficiency and developmental competence of bovine oocytes.

Prostaglandin E2 affects in vitro maturation of bovine oocytes.

The role of prostaglandin E2 (PGE2) in the successful resumption of oocyte meiosis and cumulus expansion has been well-documented. However, there remains very little information available on the influence of PGE2 on other processes that occur during oocyte maturation. In this study, we supplemented a maturation medium with PGE2 and monitored oocyte quality markers, glucose metabolism, mitochondrial status, oxidative stress, and apoptosis in the cumulus-oocyte complexes (COCs), using a well-established in vitro model of embryo production in cattle. We found that this increased availability of PGE2 during maturation led to an increase in the expression of genes associated with oocyte competence and improved the quality of blastocysts produced. Prostaglandin E2 also appeared to stimulate glucose uptake and lactate production in the COCs, both influencing the expression of enzymes involved in glycolysis and the hexosamine biosynthetic pathway. We found that PGE2 reduced intracellular reactive oxygen species levels, and simultaneously increased glutathione concentration and stimulated antioxidant gene expression in the oocyte. These results indicate that PGE2 has an important role in the protection of oocytes against oxidative stress. Mitochondrial membrane potential was also improved in PGE2-treated oocytes, and there was a reduction in the occurrence of apoptosis in the COCs. Promotion of an anti-apoptotic balance in transcription of genes involved in apoptosis was present in both oocytes and the cumulus cells. In summary, PGE2 could represent a novel autocrine/paracrine player in the mechanisms that can facilitate successful oocyte maturation and oocyte survival in the cow.