Extraction And Identification Of Exosomes From Bovine Uterine Fluid In Estrus And Effects On Development Of Ivf Embryo

Exosomes is a kind of nano-vesiclereleased from cells to outside environment by endocytosis and exocytosis,containing important biological message substances such as protein,lipids,mRNA and microRNA.The small vesicle widely involved in physiological or pathological biological activities as a "messenger".More and more studies have shown that exosomes is present in almost all of the body fluids,implying the importance and necessity of exosomes in biological activities.Evidence haveconvinced that exosomes cound play a very important role at the beginning of mammalian life – embryos.In the present study,wefirstlyextracted and identified the exosomes from the bovine uterine fluidinestrus,and then added the exosomesinto IVF medium to observe the effect on the development of IVF embryos.The study achieved results as follows:1.The bovine uterus in estrus wasrinsed by saline through catheterwith 2 mLeach time and repeated 6-8 times,andthe fluid rinsed was collected into centrifuge tube.2.Higher purity of the exosomes from uterine fluid was obtained by modified ultracentrifugation.3.The diameter of exosomes was about 100 nm with the morphology ofregular rounded or ellipticunder the transmission electron microscope.4.Immunofluorescence staining of exosomes pecific membrane protein revealed CD9+,Thewestern blot result confirmed the expression of CD9.5.To evaluate its effects on development of embryos,extracted exosomes was added to IVF embryo culture medium.The results showed that there was no significant difference between the exosomes supplementing group and the control group in the cleavage rate(81.2%vs79.1%)and the blastocyst rate(33.3%vs32.5%);6.The apoptotic cells of blastocyst were stained and counted.The results showed that there was no significant difference between the exosomes supplementing group and the control group in cellsnumberin blastocysts(110.21 ± 4.52 vs 113.11 ± 3.20),but the number of apoptotic cells was significantly lower in exosomes supplementing group than that of control group(2.68±0.30 vs 5.83±0.44,P<0.05).In summary,exosomes from bovine uterine fluid in estruscould decrease the proportion of apoptotic cells in blastocysts in vitro.