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 effects of sub-chronic arsenic poisoning on hyaluronidase activity of rat sperm.Methods Forty Sprague-Dawley (SD) rats in cleanliness grade,weighing 100-200 g,were randomly divided into four groups:control group,low-dose,middle-dose and high-dose arsenic exposure groups,10 in each group,and allotted to cages.Rats of arsenic groups were exposed to arsenic through drinking water (As2O3 was dissolved in distilled water).The concentrations of As2O3 in low-dose,middle-dose and high-dose groups were 25,50,and 100 mg/L,respectively.Body weight of each rat was weighed once a week.The rats in control group drank distilled water freely.All rats were fed for three months.After the experiment,all rats were killed and their sperms were collected.The positive rates and hyaluronidase activity intensities of the sperms in different groups were examined by the mature improved fixed-substrate film method.Results The positive rate of sperm hyaluronidase in control,low-dose,middle-dose and high-dose groups were (90.8 ± 13.2)％,(79.1 ± 15.6)％,(59.7 ± 20.3)％ and (33.8 ± 14.5)％,respectively.Compared with the control group,the positive rates of the middledose and the high-dose groups were significantly reduced(all P ＜ 0.05).The activity intensities of hyaluronidase in the sperms in control,low-dose,middle-dose and high-dose groups were (45.1 ± 24.0),(31.1 ± 21.3),(19.4 ± 15.3) and (9.8 ± 3.1)μm,respectively.Compared with the control group,the activity intensities of hyaluronidase of the middle-dose and the high-dose groups were significantly reduced(all P ＜ 0.05).Conclusion Sub-chronic arsenic poisoning reduces the activity intensities and positive rates of sperm hyaluronidase of rat,resulting in reduced sperm fertilizing capacity.