Study On The Factors Affecting The Absorption Of Awine ST Cell Exosomes

Swine Sertoli cells(ST)provide nutritional support for porcine spermatogenesis,and the influence and regulation of external environmental factors on ST cells will also affect sperm development.Exosomes are an important intercellular information transmission pathway found in recent years,which can transmit the information of upstream cells to downstream cells.Therefore,this study will help to further reveal the mechanism of Min pigs’ adaptation to the cold environment by exploring the regulatory effect of environmental factors on ST cells and the factors affecting the intercellular information transmission of ST cells through exosomes.It will help to further reveal the mechanism of Min pigs’ adaptation to cold environment.In this study,1)vesicles secreted by ST cells were extracted to identify whether they were exosomes,and the mi RNAs in them were sequenced and analyzed to obtain epigenetic-related mi RNAs.2)the exosomes secreted by donor ST cells were extracted,these exosomes were stained and incubated with recipient ST cells.The cytoskeleton status,apoptosis,and green fluorescence intensity of the cell surface of the recipient ST cells were observed,at treated with different temperatures,different culture times,and adding cytochalasin D(Cyt D)and FTY720.3)The exosomes were extracted under overexpression/inhibition of mi R-148a-3p.After the exosomes were incubated with the recipient ST cells,the q PCR method was used t o detect the relative expression of mi R-148a-3p in the recipient ST cells.4)The gene regulation function of mi R-148a-3p was verified.The results show that:(1)The vesicles secreted by ST cells have a monolayer membrane structure,hemispherical shape and a concave surface on one side,with a size range is 60-100 nm,and CD63 immunological signal is positive.These vesicles can be determined to be exosomes.(2)Ambient temperature affects mi RNA expression in exosomes,and differences in mi RNA include mi R-148a-3p related to epigenetic and energy metabolism.Therefore,mi R-148a-3p was used as the marker of regulatory signal transmission between cells and exosomes in subsequent experiments.(3)With the extension of coincubation time,the green fluorescence intensity of exosomes on the cell surface increased gradually;The green fluorescence intensity of cell surface exosomes increased gradually with the increase of coincubation temperature;Cyt D can destroy the cytoskeleton structure,and the green fluorescence intensity on the surface of receptor cells decreases with the increase of Cyt D concentration;With the increase of FTY720 concentration,the cytoskeleton morphology did not change significantly,but the apoptosis rate increased,and the green fluorescence intensity on the surface of recipient porcine ST cells decreased;The expression of mi R-148a-3p in exosomes was consistent with that in donor cells;The relative expression of mi R-148a-3p in recipient cells increased with the extension of coincubation time and the increase of coincubation temperature;The relative expression of mi R-148a-3p decreased with the increase of Cyt D concentration and FTY720 concentration.(4)mir-148a-3p can inhibit the expression of epigenetic related gene DNMT1 and metabolism related genes such as BRCA1,PCNA and PGC1.In conclusion,the results showed that ambient temperature could change the expression of mi R-148a-3p in the exosomes secreted by ST cells;At the same time,the ability of ST cells to absorb exosomes and mi R-148a-3p carried in exosomes is affected by many factors,such as temperature,time,Cty D and FTY720.mi R-148a-3p can inhibit and regulate some epigenetic and metabolic related genes.Therefore,the change of mi RNA expression in ST cells caused by the change of ambient temperature can be transmitted between cells through exosomes,but the transmission efficiency is affected by many factors.