Function And Expression Of SLO Family During Spermatogenesis In Eriocheir Sinensis

Spermatogenesis,a complex cell event,is completed in the seminiferous tubules.To make this complex process orderly,it is necessary for the intracellular and extracellular signals and membrane protein of seminiferous tubules to regulate this step strictly and accurately.The changes of membrane potential of germ cells may be closely related to spermatogenesis.In order to obtain fertilization ability,mature sperm need to experience the following phases: epididymis maturation,sperm capacitation,hyperpolarization,sperm tendency,and finally sperm acrosome reaction.These processes are related to the changes of the intracellular and extracellular concentration of ions.The ion channel of sperm plays a key role in regulating its ion balance and important physiological processes(sperm motility,acrosome reaction,tendency to egg,etc.),and the change of expression level or function of ion channel directly affects the male fertility of human and other animals.The life cycle of Eriocheir sinensis is complex,and its fertilization process and individual living environment is much different from other species.Reproductive migration is a very important physiological phenomenon in the crab.The ion concentration of fresh water is much different from that of sea water,so the crab experience a strong ion concentration change in the process of reproductive migration.Therefore,the change of ion concentration plays an important role in the gonad development and reproduction of E.sinensis.Potassium channels are the ion channels with the most types and subtypes so far.It has been reported that potassium channels can regulate sperm formation and fertilization.The SLO family is a very special potassium channel.The characteristics of such channels are not only voltage dependent,but also sensitive to the concentration of other ions,such as calcium ionone,sodium ion,chloride ion,pH,etc.Western blot,immunohistochemistry,immunofluorescence,PCR and gene clone were used to localize SLO family proteins in this paper.Then,the electrophysiological experiments of spermatogenic cells were carried out with patch clamp technique to observe the changes of SLO channel in spermatogenic cells.The results are as follows:1.Western blot assays showed that SLO1 existed in the testis and other tissues of E.sinensis,but the SLO1 protein of sperm in the seminal vesicles was less expressed.SLO2 mainly existed in spermatogenic cells in the testis and sperm in the seminal vesicles.The expression of SLO2 channel proteins had divergence between spermatogenic cells and sperm.2.SLO nucleotide fragments from E.sinensis were cloned and sequenced.The cDNA clones of SLO1 appeared to contain higher homology sequence with other species,which showed that SLO1 gene is highly conservative and it is most closely related to slo1 of Scylla pseudocerata.Although the cDNA clones of SLO2.2 have genetic similarity with other species,it did not show highly conservative characteristics.The results of phylogenetic tree analysis showed that SLO 2.2 of E.sinensis is the most similar to SLO2.2 of Cancer borealis.3.SLO1-mediated calcium activated potassium channel and SLO2-mediated sodium activated potassium channel were explored by patch clamp technique.The potassium current in spermatogenic cells of E.sinensis contained activated potassium current.Subsequently,different blockers IbTX and CdCl2 were added to observe the current.The results showed that these inhibitors could block the potassium currents.According to the blocking results,SLO1 protein was mainly expressed in spermatogonia and spermatocytes,which was consistent with the results of immunolocalization of SLO1 protein in preliminary research.SLO2-mediated sodium activated potassium channel also was expressed in spermatogenic cells.However,spermatogenic cells had different dependence on sodium ions.Spermatozoa had strong dependence on sodium ions,while spermatogonia,spermatocytes and spermatids had weak dependence on sodium ions.This indicated that the expression of SLO2 in spermatogenic cells was different.4.The effect of SLO1 channel inhibitor on acrosome reaction of E.sinensis was observed.It was found that potassium channel was not only related to the formation of sperm,but also indirectly regulated acrosome reaction of sperm.SLO1 was related to acrosome reaction.Different inhibitors exhibited different inhibition on acrosome teaction.Meanwhile,it was found that there was no direct relationship between sodium ion and acrosome reaction,which indirectly indicated that the effect of SLO2 might be more on sperm motility and capacitation.In conclusion,SLO1-mediated calcium activated potassium currents and SLO2-mediated sodium activated potassium currents were all existed in spermatogenic cells of E.sinensis.The concentration of SLO1-mediated calcium activated potassium currents tended to decline with the development of spermatogenic cells.While SLO2-mediated sodium activated potassium currents appeared less sensitive to sodion in sperm than other type of spermatogenic cells,which may be related with the spawning migration of E.sinensis.The acrosome reaction induced experiments showed that SLO1 was not only related to spermatogenesis,but also indirectly regulated acrosome reaction of spermatozoa.The closing of SLO1 channel led to the decrease of acrosome reaction ratio.SLO2 maybe have more impact on sperm motility and capacitation,which need to be verified by further experiments.