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-SION:Silencing of P34H significantly inhibits the percentage of P 34H positive rate and the activity of hyaluronidase in mouse sperm.
ABSTRACT
Objective: To investigate the correlation between the level of P34H expression and the activity of hyaluronidase in human spermatozoa. Methods: Eighty eight semen samples were collected, 68 cases were in the infertile group, and 20 in the normal control group. Semen routine analysis was referred to the WHO standard method. According to the difference of semen parameters, 68 cases of infertile males were divided into the infertile group with normal semen parameters and the abnormal semen parameters. Western blotting was used to detect the level of P34H expression on spermatozoa. The P34H-positive rate on human spermatozoa was determined by indirect immunofluorescent staining using anti-P34H antibody. The HYD-positive rate and HYD-activity intensity in all samples were examined by improved fixedsubstrate film method. Results: The level of P34H protein expression and the percentage of the P34H-positive rate in infertile groups with normal semen parameters and abnormal semen parameters were significantly lower than that in the control group (P<0.05). The activity of HYD (HYD-positive rate, HYD-activity intensity) in the infertile groups with normal semen parameters and abnormal semen parameters were also significantly lower than that in control group (P < 0.05). The relation between the P34H protein expression and HYD-positive rate, HYD-activity intensity had a significant positive correlation (r = 0. 449, 0. 431; P < 0.01); the relation between the the percentage of P34H-positive rate and HYD-positive rate, HYD-activity intensity had a significant positive correlation (r=0.727, 0.691;P <0.01). Conclusion: The level of P34H protein expression and the percentage of the P34H-positive rate are decreased, while the activity of HYD (HYD-positive rate, HYD-activity intensity) is reduced in male infertility.