Study Of Effect And Mechanism Of OMVs Of Proteus Mirabilis On Boar Sperm

There’s a severely decreased on quality of boar semen by bacterial contamination.And the mechanism of this phenomenon is mostly focused on the oxidative stress or apoptosis of sperm induced by bacterial metabolites.And the metabolites are mainly toxin factors.All Gram-negative bacteria and some gram-positive bacteria could release extracellular vesicles.These vesicles is not only a way of communication between bacteria,also for carrying toxin invasion.And could the extracellular vesicles from bacteria change the characters of sperm?There is no report about it.So in order to research this problem,extracellular vesicles is acquired from the bacteria which isolated from boar semen in vitro,and the semen quality is detected after the vesicles treatment.The results are as follows:（1）6 bacterias were isolated from semen of six guanzhong-black boar:Proteus mirabilis,Pseudomonas spp,Bacillus fusiformis,Escherichia,Acinetobacter junii,Bacillus cereus.And Proteus mirabilis was chose to acquire vesicles.Proteus mirabilis is Gram-negative bacteria and the extracellular vesicles from negative bacteria is called outer membrane vesicles（OMVs）.OMVs are spherical,bilayered membrane nanostructures with an average diameter of 20-250nm.The Proteus mirabilis reached a stable stage for about 9 hours according to the growth curve.So vesicles were collected after 9 hours.The OMVs had a diameter of about 103nm（70-200 nm）,and it was at the concentration of 5.85×10¹¹/mL.（2）The boar semen was treatment with a series of concentration gradients of OMVs（0.25μg,0.5μg,2.50μg,5.00μg,10.00μg,20.00μg,40.00μg）of Proteus mirabilis.The result showed that there was a decrease on sperm total motility and progressive motility after OMVs treatment.And there was a decline of plasma membrane integrity and acrosome integrity after OMVs treatment too.So the OMVs of Proteus mirabilis caused a decrease on semen quality.In order to investigate sperm fertility after OMVs treatment,the zona pellucid binding assay was carried out.The result showed that the number of sperm binding to zona pellucida was decreased after the OMVs treatment after just one day.（3）The contents of reactive oxygen species（ROS）and malonaldehyde（MDA）were increased after OMVs treatment.And there was disorder of Total Antioxigenic Capacity（T-AOC）of sperm.These indicated OMVs of Proteus mirabilis increased oxidative stress of sperm.（4）To further explore the mechanism of OMVs on sperm,DiI labeled OMVs were used.DiI-OMVs were incubated with sperm and the result indicated the OMVs could bind to the middle piece of sperm.And the scanning electron microscope also proved OMVs could bind to sperm.But the mechanism of effect on sperm of OMVs still needs further exploration.And the result of LPS-Beads revealed there are many proteins that could bind to LPS in sperm.OMVs may influence on sperm by bind to the proteins.In short,OMVs influenced sperm by binding to sperm membrane.