The Effects Of MEK Inhibitor And Knock-down AR On The Expression Of GBD104a In Epididymal Caput Cells Of Buck

The goat P-defensin 104a(gBD 104a)was located in the epididymal caput epithelial cells of buck,acrosome and middle mitochondria of sperm.It may play important roles in sperm maturation and capacitation.However,the expression regulation mechanism of gBD104a was unclear.Therefore,the objective of this study was to investigate the effect of MEK inhibitor and knock-down AR gene on the expression of gBD104a in epididymal caput cells of buck.Epididymal caput of kid buck was isolated,digested by trypsine,cultivated for 48 h.Epididymal caput cells was passaged the forth or fifth generations,then detected.The numbers of the live cell and cell apoptosis were determined by Flow cytometer after 48 hours with addition of different concentration of 17 β-estradiol(E2,0.001 ng/mL,0.01 ng/mL,0.1 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL),dihydrotestosterone(DHT,10 ng/mL,20 ng/mL,40 ng/mL,80 ng/mL,160 ng/mL,320 ng/mL)and MEK signal pathway inhibitor PD0325901(1.0 μM,2.5 μM,5.0 μM,10.0 pM)in the cultural medium,respectively.At the same time the cell were collected and examined the expression of gBD104a by QRT-PCR and Western blotting.Three AR knockout vectors were designed by CRISPR/Cas9 system and determined by QRT-PCR and Western blotting.The expression of gBD104a was detected after knock-down AR gene.The results of the study were as follows:(1)After 48 hours with adding 0.1 ng/mL E2 in culture medium,the expression of gBD104a mRNA in epididymal caput cells of buck was significantly higher than that in the control and the group with addition of 100 ng/mL E2(P<0.05).After 48 hours with adding 160 ng/mL DHT in culture medium,the expression of gBD104a mRNA in epididymal caput cells of buck was significantly higher than that in the control and the groups with addition of 10 ng/mL,20 ng/mL,40 ng/mL DHT,respectively(P<0.05).(2)After adding 0.1 ng/mL E2 and the different concentration of PD0325901 in culture medium respectively,the expression of gBD104a in epididymal caput cells of buck was significantly lower than that in the group with addition of 0.1 ng/mL E2.And adding 0.1 ng/mL E2 and 1.0 μM PD0325901 in culture medium,the expression of gBD104a was significantly lower than that in the control(P<0.05).After adding 160 ng/mL DHT and 5.0 μM PD0325901,the expression of gBD104a in epididymal caput cells of buck was significantly higher than that in other groups(P<0.05).(3)After adding 0.1 ng/mL E2 and the different concentration PD0325901 in culture medium,respectively,the percent of viable cells was significantly lower than that in the group with addition of 0.1 ng/mL E2.The percent of apoptotic cells was higher than that in the control and the group with addition of 0.1 ng/mL E2 in culture medium(P<0.05).While adding 160 ng/mL DHT and the different concentration of PD0325901 in culture medium respectively,the percent of viable cells was significantly lower than that in the control and the group with addition of 160 ng/mL DHT(P<0.05)and the percent of apoptotic cells was significantly higher than that in the group with addition of 160 ng/mL DHT(P<0.05).(4)The plasmids of pCas9/gRNAl-AR were delivered into the epididymal caput cells of buck.pCas9/gRNAl-AR-1452 was significantly reduced the expression of AR.After knock-down AR of the epididymal caput cells with pCas9/gRNAl-AR-1452,the expression of gBD104a mRNA and gBD104a was significantly decreased than that in the control(P<0.05).In conclusion,after adding E2 and DHT,the expression of gBD104a in epididymal caput cells of buck was promoted.E2 and DHT could regulate the expression of gBD104a by MEK signal pathway,and DHT could enhance the expression of gBD104a by androgen-androgen receptor signal pathway.