Mechanisms Of Toll-like Receptor In Affecting Mouse Sperm Motility

Infections of genital tract are one of the most serious causes of infertility in males and females, and infections can initiate the immune response. Toll-like receptors (TLRs), which function as vital molecules in innate immune system, play essential roles in protecting organism from external inference.TLRs have recently been found to be expressed in sperm. Sperm motility has been considered as an important process for successful fertilization. To study the role of TLRs in sperm function, we firstly examine the effects of TLR agonists on sperm motility. Surprisingly, the TLR agonists are capable of inhibiting sperm motility. In this study, the specific signaling pathway of TLR in influencing sperm motility will be investigated. Furthermore, we will explore the concrete biologic events conducted by TLR signaling in suppressing sperm motility.Our study was divided into three parts:Part I. Influence of different kinds of TLR agonists on sperm functionObjective:To observe the effect of TLR agonists from different kinds of pathogens on sperm motility and to explore its possible reason for inhibiting sperm motility.Methods:RT-PCR was used to analyze the TLR expression in mouse sperm. The influence of TLR agonists on mouse or human sperm motility was measured using Computer-Aided-Sperm-Analysis (CASA). Flow cytometry was performed to check the effects of TLR agonists on mouse sperm apoptosis.Results:All TLRs were expressed in mouse sperm except TLR3, which was rarely expressed in sperm. When the mouse sperm was treated with TLR agonists for6h, most of the TLR agonists exhibited significant inhibition in sperm motility except the agonist of TLR3. Moreever, TLR agonists could also inhibit the normal human sperm motility in a time-dependent manner. Zymosan or LPS could only exhibit inhibition when incubated with human sperm for6h, but R848initiated the inhibition of sperm motility at2h. Finally, TLR agonists could not efficiently promote the sperm apoptosis compared with the group treated with saline.Conclusion:TLR agonists could effectively decrease mouse or human sperm motility. The inhibition of sperm motility was not induced by sperm apoptosis. Part Ⅱ. Study of the TLR signaling pathway in affecting sperm motilityObjective:To investigate the exact signaling pathway of TLR in affecting sperm motility.Methods:MyD88-/-mice were used to study the role of MyD88-dependent pathway in TLR agonist-inhibiting sperm motility. Sperm was treated with LPS for2,4and6hours and proteins were then extracted. The effect of TLR agonists on the phosphorylation level of sperm proteins such as IRAK, PI3K, GSK3a, AKT, GSK3β, ERK and p65, were examined through Western-blotting. Moreover, mouse sperm was treated with TLR agonists for6h after inhibitors of the IRAK, PI3K, MEK/ERK or NF-κB pathway were executed on sperm.The effect on sperm motility was assayed by CAS A. The influence of the PI3K inhibitor on the phosphorylation of AKT and GSK3a was detected via Western-blotting.Results:TLR agonists showed no effect on the sperm motility of MyD88-/-mouse. TLR agonists resulted in the significant phosphorylation of IRAK, PI3K or GSK3a but not in the phosphorylation of AKT, GSK3β,ERK or p65. Signaling blockade by IRAK or PI3K inhibitors rescued TLR agonist reduced sperm motility, but not for either MEK/ERK or NF-κB inhibitors. Furthermore, the phosphorylation of GSK3a could be inhibited by the PI3K signaling blockade, but that of AKT was unaffected by PI3K inhibitor.Conclusion:TLR signaling decreased sperm motility in a MyD88/IRAK4/PI3K/GSK3a-dependent but not AKT-dependent manner. Part Ⅲ. Study of the TLR signaling in affecting sperm biological processObjective:To examine the changes in biological process induced by TLR signaling that eventually resulted in decreased sperm motility. Moreover, the function of exact TLR signaling pathway performed in these changes will be investigated.Methods:Luciferase reaction was used to detect changes in ATP level of mouse sperm, which was incubated with TLR agonists for6h. Moreover, the effect of TLR agonists on the ATP level of MyD88-/-mouse sperm was assayed via a similar method. F1Fo ATPase ELISA assay kit was used for the determination of the human sperm F1Fo ATPase activity affected by TLR agonists for6h. The mitochondrial membrane potential changes of sperm were detected using a JC-1assay kit via flow cytometry. The molecules translocated into mitochondria were observed via western blotting.Results:TLR could significantly decrease the ATP level of wild-type mouse sperm compared with control, but no change was observed on the ATP level of MyD88-/-or pik3cd-/-mouse sperm. A remarkable increase was noted in the F1Fo ATPase activity after TLR agonists were incubated with human sperm for6h. The mitochondrial membrane potential of mouse sperm decreased in a time-dependent manner. Predominantly, a significant decrease in mitochondrial membrane potential was noted at4h. TLR agonists promoted the molecules translocation into mitochondria.Conclusion:TLR signaling inhibited sperm motility via indirect ATP consumption, which was caused by a decrease in mitochondria membrane potential and this process is MyD88and PI3K dependent.