A simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore-treated sperm using the sperm-adhesion treatment was significantly higher (P less than 0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 offspring (45.8%). The inseminations performed with the sperm-adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore-treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore-treated sperm can be improved by applying the sperm-adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.
The effect of calcium concentration in culture medium on the fertilization of subzonally microinseminated mouse oocytes was examined. Oocytes were injected with a single spermatozoon so that the sperm head was forced to adhere onto the ooplasmic membrane with a micromanipulation technique. For the inseminations, epididymal spermatozoa preincubated in culture medium and those treated with ionophore A23187 were used. Inseminated oocytes were cultured using media with three different calcium concentrations of 1.71, 3.42, and 5.13 mM; 40.0%, 71.6%, and 47.9% of oocytes microinjected with preincubated sperm were fertilized after incubation with those media, respectively. When the oocytes inseminated with ionophore-treated sperm were incubated in media containing 1.71 and 3.42 mM calcium, their fertilization rates were 58.2% and 87.5%. Thus fertility of subzonally microinseminated oocytes was obviously enhanced when cultured in medium with 3.42 mM of calcium, irrespective of being inseminated with preincubated sperm (P < 0.01) or with ionophore-treated sperm (P < 0.005). Some of the microinseminations with preincubated sperm were performed without sperm adhered to the oolemma. In these cases, the incidence of fertilization was not improved by incubating the inseminated oocytes in medium containing 3.42 mM calcium (32.6%) as compared to those incubated in medium with 1.71 mM calcium (28.3%). These results suggest that the concentration of extracelluar calcium exerts an important effect on the progress of fertilization events subsequent to sperm adherence onto the ooplasmic membrane. Almost 80% of the zygotes fertilized via incubation in medium with 3.42 mM of calcium developed into blastocysts after culturing in vitro.
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