The Application Of Chemical Activation On Spermatogenic Cells Of Mouse In Vitro Fertilization

Part Ⅰ The research of mouse haploid spermatidsmicroinjecting methodologyObjective：To explore the application values of micromanipulation system with themodified holding pipette on mouse spermatids in vitro fertilization.Methods：The routine holding pipette was remodeled into a wide trumpet-shapedopening by repeatedly calcination, and then round spermatids were injected into mousemature oocytes by a special manuduction. The survival rate of mouse oocytes injectedwith modified holding pipette was counted after ROSI30minute and compared with thetraditional one with a narrow opening.Results：Round spermatids were injected into320mouse oocytes for each holdingpipette，the survival rate of mouse oocyte after ROSI by modified holding pipette wassignificantly higher than routine one.Conclusion：Modified holding pipette，easy to make up and manipulate，no needingpiezo-actuated micromanipulation system，has a great application value in research ofmouse in vitro fertilization. Part Ⅱ The application of chemical activation on mouseround spermatids in vitro fertilizationObjective： To explore the optimal chemical activation protocol by comparing theactivation effects of oocytes with single chemical activator and two combined chemicalactivators following microinjection with spermatids from spermatogenic cells in vitroculture.Methods: Six chemicals such as ethanol, calcium ionophore A23187, ionomycin,strontium chloride, cycloheximide and6-dimethylaminopurine were selected asactivators. Following round spermatid injection (ROSI), oocytes were activated by allsix different concentration activators at different times, and then rates of normalfertilization, cleavage, morula and blastocyst were observed and compared. Accordingto the different activation channels as well as the optimal concentration and action timesof activators, oocytes after ROSI were activated by eight combination protocols, theactivation effects of different combination protocols were evaluate with the rates ofnormal fertilization, cleavage, morula and blastocyst. Round spermatids from adultmouse testis were employed as a control and compared with the one from in vitroculture.Results:(1)For the single activation, each optimal protocol as followings:①7%ethanol for6min;②5μmol/LCIA for5min;③5μmol/L Ion for5min;④2mmol/L6-DMAP for2h;⑤10mmol/L SrCl2for1.5h;⑥10μg/mL CHX for1.5h, of those10mmol/L SrCl2for1.5h showed the best activation effects, but there was no significantdifference in activation effects among all groups except CHX.(2)For the combinationactivation, the morula and blastocyst rate (29.63%) in ethanol+6-DMAP group was significantly higher than other groups and all single activators except SrCl2(P<0.05),therefore, it would be considered the optimal activation protocol.(3) Following adultmouse testis round spermatid injection, oocytes were activated by ethanol+6-DMAP,there were no significant difference in rates of normal fertilization, cleavage, morulaand blastocyst compared with round sprematids from spermatogenic cells in vitroculture.Conclusion:10mmol/L SrCl2for1.5h and ethanol (7%,6min) combined6-DMAP (2mmol/L,2h) might be served as the optimal protocols for single and combinationchemical activation respectively. The fertilization and embryo develepment potential ofround spermatids from adult mouse testis after ROSI showed no significant differencewith the spermatids from in vitro culture.