| Sperm hyperactivation is a prerequisite for its capacitition and its involvement in acrosome reaction. It provides forceful thrust needed to free the sperm cell from the capture of oviduct, propelling them through mucus in the oviductal lumen and enabling them to penetrate the cumulus and zona pellucida surrounding the egg. Researches including our previous studies have showed that the CatSper2protein is one of the vital channel proteins which mediate Ca2+influx in the course of sperm hyperactivation, and it expressed exclusively in the testis and the mature sperms. In the recent years, relative researches were focused on the biologic function of CatSper2protein, however, the information about its role on male fertility regulation is limited. It can predict that inhabiting one of the submit expression of targeted CatSper2as the site for fertility mancipation will be the next focus of associated studies.In this study, one the submit of CatSper proteins was chosen as the targeted site, small interfering RNA plasmid vector was constructed and synthesized, in vivo electroporation (EP) and rete testis microinjection-mediated plasmid were adopted to silence CatSper2expression, ultimately block the extracullar Ca2+which is essential in sperm hyperactivation. Specifically, the techniques of in vivo bioluminescence imaging, fluo-anatomical lens, FCM, CASA, Q-PCR, Western-blot, confocal microscopy, IVF as well as histological analysis were used to detect the transfection efficacy of the testis and the sperms in epididymis, the sperm hyperactive motility, inhibition effect on CatSper2, intracullar Ca2+influx, sperm fertility the survival rate, and the effect of in vivo EP and testis microinjection-mediated plasmid on the function of both the testis and epididymis.The main results were as following:1. Successful construction and synthesis of small interfering RNA plasmid vector and virus vector for CatSper2, and high transfection efficacy in vivo in rete testis The optimal interference site was screened and chosen as the target in the construction of plasmid vector and virus vector for CatSper2, then rete testis microinjection and in vivo EP were adopted in this study, high transfection efficacy and expression were achieved. Compared to the consistent expression of virus vector, the method of plasmid vector combined with in vivo EP was showed to be more effective to silence CatSper2. In contrast, in spite of the relative higher rate of positive sperm was found in the group transfected with plasmid vector than the one with virus vector, which was87.75%to83.3%(P>0.05), it was found that after60days of transfection, the positive sperm rate of the virus vector group (78.7%) was moderately higher than that of the plasmid group (77.75%)(P>0.05) Furthermore, the inhibit effect of virus vector maintained longer than that of the plasmid vector. It was suggested that the virus vector could be more effective to conform into the host cell. But the difference of the results bare no meanings statically.2. the efficacies of transfection and expression of plasmid vector and virus vector to the testis were both dose and time-dependent.According to the analysis of the BLI signal data and fluo-anatomical lens, as the amount of injected plasmid and virus vector increased respectively from25-45μg and10μl～40μl, the acquired GFP signal form the testis was also turned to be more strong, which demonstrated this was dose-dependent. And the strongest signal was acquired when the injected amounts were50μg and40μl respectively. But there was no statistically significant difference between the two groups that with45μg and50μg plasmid vector (P>0.05). Meanwhile, no significant difference was found between the two groups that with35μl and40μl virus vector. Therefore, the injected amount of45μg plasmid vector and35μl virus vector were followed in the subsequent experiment. When the some amount injection were carried out, the results showed that the efficacies of transfection and expression of plasmid vector and virus vector to the testis were time-dependent. The testicular fluorescence signal peaked on the13d and the22d respectively.3. Hyperactivation triggered by intracellular Ca2+and sustained by exteracellular Ca2+Sperms were incubated for1min in TALP with a50μM of Thimerosal but free of Ca2+. VCL, ALH and BCF detected by CAS A were listed in Table3. The results indicated more than90%of hyperactivated sperms in the control and treated groups. This was significantly higher than the normal sperms whose hyperactivation rate was only5-10%. Subsequently, sperms were incubated for5min in capacitation with procaine at a final concentration of5mM. CASA indicated that the hyperactivation rate of treated sperms dropped to11.1%. The swimming pattern of the sperms was featured by helical motility with low amplitude and high frequency of flagellar beating, as previously reported. However, the control group maintained high hyperactivation rate of95.7%(P<0.05)(Table3) for a longer time if no blocking agents were added.3. According to the CASA analysis, sufficient Ca2+ion in intracellular Ca2+stores of sperms could trigger hyperactivation, but the hyperactiation process could not be sustained without extracellular Ca2+influx as supplement.When sperms were incubated in a50μM of Thimerosal with or without extracellular Ca2+, more than90%of sperms in the control and treated groups were hyperactivated. Nevertheless, the hyperactivation activity lasted only a very short time. After that, when the treated sperms were incubated for5min in the5mM procaine, the concentration of the cytoplasm Ca2+still remained at a low level, because no more CatSper2on the plasma membrane could be activated by procaine, therefore, no more extracellular Ca2+could enter into the cytoplasm. It was speculated that sufficient Ca2+ion in intracellular Ca2+stores of sperms could trigger hyperactivation, but because of the limited amount of intracellular Ca2+ion in sperms, the hyperactiation process could not be sustained without extracellular Ca2+influx as supplement.4. The transfected sperm failed to penetrate the zona pellucida of intact eggs due to the lack of intracellular Ca2+peakThe results of the fertility essay showed that treated spermatozoa failed to penetrate the zona pellucida of intact eggs. In contrast, they could fertilize the eggs with removal of the extracellular matrix by0.4%hyaluronidase. And no difference was found between the two groups. To elucidate the that why the treated sperms failed to fertilize the normal eggs, here we analyzed the effects of elevating the viscosity of the medium on the motility of control and treated spermatozoa. We found that, though the mobility of all sperms was confined in the higher-viscosity media, compared with the total loss of the forward mobility of the treated group, the sperms of the control group still possessed48%mobility. It was suggested that CatSper2was essential for the generation of hyperactivated form of motility which was necessary for successful fertilization. Removal of the extracellular matrix corrected the infertility of treated sperm cells, suggesting that the zona pellucida of the egg represented an absolute barrier to CatSper2spermatozoa17. Compared to the treated group, we found that treated spermatozoa failed to penetrate the zona pellucida of intact eggs. This finding suggested that despite normal forward velocity and percentage of motile cells, To further verify the change of fertilization capability of the treated sperms, we tested the effects of elevating the viscosity of the medium on the motility of control and treated spermatozoa. We found that, the virus vector group totally loss the forward mobility, and the sperms of the control group still possessed48%mobility. This demonstrated that the main reason for the failure of fertilization was that the hyperactivated mobility of treated sperms was confined in the higher-viscosity media.5. Histological analysis and the sperm survival rate essay showed that the no significant side effect was caused by microinjection of DNA plasmid and EP on the function of rete testis and spermsThe methods of microinjection of DNA plasmid and EP were adopted to inhibit the expression of CatSper2, by which ultimately as a way to manipulate the male fertilization. However, the key point was to ensure the low side effect caused by the techniques on the tissue and cells. Here we observed the function of testis tissue and the duration of sperm survival. According to Fig.4-6, GFP was expressed consistently in epithelial cell of seminiferous tubules in different developmental statues. And no damage or petechia was found on the tissue. This verified that these methods imposed low side effect on spermatogenesis. Additionally, according to Fig.4-5, the in vitro survival rate of the treated sperms was not significantly effected compared to the control group.CatSper protein family plays an important role in sperm hyperactivation and fertilizing capacity. For the spermatozoa, abnormal expression of CatSper protein subunits will not only lead to failure in the acquirement of hyperactivate activation, but also in the capability of penetrating the zona pellucida. In the present study the CatSper2expression was suppressed by microinjection of plasmid DAN into the rete testis combined with EP, which indicated that male fertility could be manipulated by knocking down the CatSper2expression in order to suppress the signal channel of sperm hyperactivation. Furthermore, microinjection of EP-mediated plasmid DNA did not affect the function of testis and the survival rate of sperm. |
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