Effects of epididymal P34H gene silencing on expression of P34H and ac-tivity of hyaluronidase in mouse sperm / 中国病理生理杂志

ABSTRACT

AIM:To investigate the effects of P34H gene silencing on the expression of P34H and activity of hyaluronidase (HYD) in mouse sperm.METHODS:The recombinant plasmid series of P34H targeted short hairpin RNA (shRNA) were constructed by GV248 plasmids vector.These recombinant plasmids were transformed into DH 5αcompetent cells, and the plasmids were taken from DNA sequencing analysis .The HEK293T cells were co-transfected with shRNA and lentiviral packaging plasmids .The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to in -ject into the mouse epididymis and the expression of P 34H at mRNA and protein levels was detected by real-time PCR and Western blot, respectively.The location of P34H protein on the mouse spermatozoa was determined by indirect immunofluo-rescent staining using P34H antibody.The positive rate and activity intensity of HYD was detected by modified sodium hya-luronate-gelatin membrane.RESULTS:DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were suc-cessfully inserted into the lentiviral vectors .P34H expression in epididymis tissue was significantly decreased at both mR-NA and protein levels compared with those of the non-transfected and normal control vectors (P<0.05).The GV-P34H-shRNA-1 played a significant role in reducing the percentage of P 34H positive rate and the activity of HYD in mouse sperm.The silencing effect did not significantly differ between the non-transfected and normal control vectors .CONCLU-SIONSilencing of P34H significantly inhibits the percentage of P 34H positive rate and the activity of hyaluronidase in mouse sperm.