Extracellular vesicles (EVs) have been identified within different body fluids and cell culture media. However, there is very little information on the secretion of these vesicles during early embryonic development. The aims of this work were first to demonstrate the secretion of extracellular vesicles by pre-implantation bovine embryos and second to identify and characterize the population of EVs secreted by bovine blastocysts during the period from day seven to nine of embryo culture and its correlation with further embryo development up to day 11. Bovine embryos were produced by in vitro fertilization (IVF) or parthenogenetic activation (PA) and cultured until blastocyst stage. Blastocyst selection was performed at day 7 post IVF/PA considering two variables: stage of development and quality of embryos. Selected blastocysts were cultured in vitro for 48 hours in groups (exp. 1) or individually (exp. 2) in SOF media depleted of exosomes. At day 9 post IVF/PA the media was collected and EVs isolated by ultracentrifugation. Transmission electron microscopy revealed the presence of heterogeneous vesicles of different sizes and population: microvesicles (MVs) and exosomes (EXs) of rounded shape, enclosed by a lipid bi-layer and ranging from 30 to 385 nm of diameter. Flow cytometry analysis allowed identifying CD63 and CD9 proteins as exosome markers. Nanoparticle tracking analysis generated a large number of variables, which required the use of multivariate statistics. The results indicated that the concentration of vesicles is higher in those blastocysts with arrested development from day 9 up to day 11 of in vitro development (6.7 x 108 particles/ml) derived from IVF (p <0.05), compared to PA blastocysts (4.7 x 108 particles/ml). Likewise, the profile (concentration and diameter) of particles secreted by embryos derived from IVF were different from those secreted by PA embryos. In conclusion, we demonstrated that bovine blastocysts secrete MVs/EXs to the culture media. Data suggest that characteristics of the population of EVs vary depending on embryo competence.

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1-5) and late luteal phases (days 13-18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD-117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin-embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c-KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86–91%) and snoRNA (9–14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

In cattle, embryos elongate before implantation and after hatching. Changes in gene expression during this transition are not well studied. Especially important are variations in the expression of pluripotency-associated genes as a result of assisted reproductive biotechnologies, such as cloning and in vitro fertilization (IVF). We hypothesize that there will be a decline in the expression of key pluripotency-associated genes and an increase in the expression of IFN-tau in elongated embryos when compared with day-7 blastocysts. To test this we generated cloned and IVF bovine day-7 blastocyst and day-17 elongated embryos (day 0 = day of nucleus transfer or IVF). Gene expression in all embryos was assessed via RT-qPCR. OCT4 was overexpressed (p < 0.05) in the cloned blastocysts when compared with IVF. No differences in gene expression at this stage between cloned and IVF embryos were found for EOMES, NANOG and FGF4. At elongation EOMES, NANOG and FGF4 were upregulated in IVF embryos (p < 0.05). IFN-tau and OCT4 were expressed at similar levels. There were changes in the expression levels for all transcripts between blastogenesis and elongation. NANOG, IFN-tau and EOMES were overexpressed in all the elongated embryos (p < 0.05), FGF4 was underexpressed in both treatments. OCT4 dropped drastically in the cloned elongated embryos, but not in the IVF. Interestingly only adult donor cells (but not fetal) from which the cloned embryos originated also expressed high levels of OCT4. Our findings might help to understand the shift of gene expression during elongation and to identify key markers of embryonic development useful for embryo screening purposes.

Stem cells have been postulated as responsible for cell regeneration in highly and continuously regenerative tissues such as the endometrium. Few studies in cattle have identified and specified the presence of stem cells in the endometrium during the oestrous cycle. The aim of this study was to investigate the presence of mesenchymal stem cells (MSCs) in the bovine endometrium during the follicular phase (FP) of the oestrous cycle. Uterine tissue was collected in the time-frame comprising day 18 of the cycle and ovulation (day 0). We isolated, cultured and expanded four primary cell lines from endometrium and identified byRT-qPCR the expression of OCT4, SOX2 but not NANOG (undifferentiated/embryonic markers), CD44 (MSCs marker) and c-KIT (stem cell marker) genes; and the encoded Oct4, Sox2 and Cd44 proteins by Western blot or immunostaining of paraffin-embedded tissue in endometrium. We demonstrated that cells isolated from bovine endometrium displayed essentially the same gene expression pattern; however, at the protein level, Oct4 and Cd44 were not detected. Besides, they showed typical functional characteristics of MSCs such as fibroblast-like morphology, plastic adherence, high proliferative capacity, clone formation in vitro and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. We obtained for the first time an extensive characterization of undifferentiated cells populations contained in the bovine endometrium during the FP of the oestrous cycle.