| Objectives: Clarification of the molecular mechanisms underlying the regulation of human spermatozoa function is crucial for the diagnosis and treatment of male infertility and the development of male contraception methods or drug. Volume regulation is important for sperm to regulate its physiological functions. The volume-sensitive chloride channels are a key determinant of the ability for cell volume regulation, and play important role in the regulation of cell volume under hypotonic conditions in healthy human sperm. Animal experiments showed that the volume regulation capacity was poor in infertile mouse sperms. However, the volume regulation capacity and the expression of chloride channels are still unclear in the sperms from the asthenozoospermia patients. The aim of this study was to detect the ability of volume regulation under hypotonic conditions, to investigate the roles of chloride channels in volume regulation and movement of sperms, and further to study the expression of volume-sensitive chloride channels and their function in the movement of the sperms from asthenozoospermia patients. This study may supply new knowledge for the treatment of asthenozoospermia patients and the development of male contraception strategies and ion channel-targeting drugs.Methods: 1) The computer-assisted semen analysis system(sperm class analyzer, CASA) was used to regularly check and analyze the collected semen, following the methods described in the book “WHO laboratory manual for the examination and processing of human semen(fifth edition, editted by World Health Organization, 2010)”. 2) The normal semen(normozoospermia) and the abnormal semen(asthenozoospermia) were obtained from the Male Laboratory and human sperm bank of Family Planning Hospital, Guangdong Province. 3) The motility of human spermatozoa was evaluated with the sperm class analyzer(CASA) system by measuring the Progressive Motility(PR), Straight-Line Velocity(VSL), Curve-Line Velocity(VCL) and Averaged Path Velocity(VAP). 4) The cell volume of normozoospermia and asthenozoospermia sperms was calculated from the Forward Scatter(FSC) signals recorded by the flow cotymetery. 5) The expression and the localization of Cl C-3 chloride channels were examined using the confocal immunofluorescent microscopy. The relative expression level of Cl C-3 was measured by the flow cytometry.Results: The cell volume of the normal sperms from the normozoospermia men was maintained stable under the isotonic solution BWW330, and was not significantly changed when bathed in the hypotonic solution BWW290, indicating that the normal sperms can efficiently activate volume regulation mechanisms under hypotonic conditions in order to restore their normal volume. However, the cell size of the sperms from the asthenozooseprm patients was increased in the hypotonic solution BWW290, indicating that the abnormal sperms cannot efficiently regulate their own cell volume under hypotonic condition. The results suggest that volume regulation mechanisms are abnormal in the sperms from the asthenozooseprm patients, leading to a weaken ability to regulate cell volume. The chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB, 100 mmol/L) and 4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid(DIDS, 100 mmol/L) significantly reduced the ability to regulate the cell volume under hypotonic conditions in the normal sperms(P <0.01), leading to an increase in cell size. In the asthenozoospermia sperm, there was no significant effect of the chloride channel blockers on cell volume under the hypotonic conditions, indicating the insensitivity of the abnormal sperm to the blockers and suggesting the abnormal expression in quantity and/or subtypes of the chloride channels in the asthenozoospermia sperm. Decrease of extracellular osmolarity to 290 m Osm/kg from 330 m Osm/kg increased the Progressive Motility(PR), Straight-Line Velocity(VSL), Curve-Line Velocity(VCL) and Averaged Path Velocity(VAP), indicating that hypotonic stimulation promotes the movement of the normal sperms. However, similar treatments significantly reduced the PR, VSL, VCL and VAP in the sperms from the asthenozoospermia patients, indicating the attenuation of motility by the hypotonic challenge in the abnormal sperms. Both the chloride channel blockers NPPB and DIDS could significantly reduce the motility of the normozoospermia and asthenozoospermia sperms in the hypotonic(BWW290) and isotonic(BWW330) conditions. Furthermore, the inhibitory efficiency of DIDS was higher than that of NPPB, and the inhibitory effect of NPPB on the normozoospermia sperm motility was stronger than on asthenozoospermia sperms in the BWW290 medium. The expression level of Cl C-3 chloride channel proteins was much higher in the normozoospermia sperms than in the asthenozoospermia sperms, detected by flow cytometry. Further study with the confocal immunofluorescent microscopy demonstrated that both the sperms expressed Cl C-3, and the Cl C-3 immuofluorescence was distributed in the neck and tail, but the expression level of Cl C-3 in the neck was significantly down-regulated in the asthenozoospermia sperms(P <0.01). The chloride channels Cl C-5 and Cl C-7 were not detectable by the confocal immunofluorescent microscopy in the normal(from normozoospermia men) and the abnormal(from asthenozoospermia patients) sperms.Conclusions: 1) The ability of cell volume regulation in the asthenozoospermia sperms was much weaker than that in the normzoospermia sperms. Chloride channels play an important role in volume regulation of the human spermatozoa. 2) Hypotonic stimulation increased the motility of the normozoospermia sperms, but attenuated the motility of the asthenozoospermia sperms. Chloride channels are important regulators of sperm mobility. 3) The expression level of Cl C-3 chloride channel proteins in the normozoospermia sperms was much higher than in the asthenozoospermia sperms, especially in the neck, suggesting that the activation of chloride channels in the sperm neck is a key factor in regulation of sperm movement. |
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