Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova

Abstract

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.

Calcium concentration and fertilization by subzonal insemination of a single spermatozoon in mouse oocytes

“…According to the results, it is thought that -70% (87.5% of 78.0%) of the normalappearing sperm population will fertilize oocytes that will develop into blastocysts. Additionally, our previous experiment (Kobayashi et al, 1992a) has revealed that -70% of mouse oocytes microinjected with a single spermatozoon under the zona pellucida were fertilized and almost one-half of the generated zygotes developed normally into offspring after implantation. Considering our data, it is conceivable that a considerable number of spermatozoa in a population may have the potential to fertilize oocytes and contribute to their further development with the aid of the micromanipulation technique.…”

“…A droplet of sperm suspension in methyl cellulose was also placed in the same well, and both droplets were covered with mineral oil. The above procedures for gamete preparations were described in detail elsewhere (Kobayashi et al, 1992a).…”

“…Space of Oocytes The procedures followed for microinjection of sperm under the zona pellucida have previously been presented by Kobayashi et al (1992a). For most of the microinseminations in this study, sperm cells were introduced under the zona pellucida so that their heads were forced to adhere onto the ooplasmic membrane as is illustrated in Figure 1.…”

“…Palermo and Steirtghem (1991) reported that microinseminated mouse oocytes were fertilized with considerable frequency (70%) when acrosome reaction of sperm was enhanced by a combination of biochemical (dbcGMP plus imidazole) and electrical stimuli. Kobayashi et al (1992a) mentioned that the fertilization of subzonally microinseminated mouse oocytes was improved by artificially assisting the adhesion of the sperm head onto the ooplasmic membrane. In that experiment, almost 70% of oocytes were fertilized when the sperm-adhesion treatment was applied to microinsemination of a spermatozoon pretreated with ionophore A23187 to induce acrosome reaction.…”

“…In the present study, we incubated subzonally microinseminated mouse oocytes in media with different Ca concentrations and examined the fertilization of those oocytes. Oocytes were microinseminated with a single spermatozoon in combination with or without a manipulation that assists adhesion of the sperm head onto the oolemma (Kobayashi et al, 1992a), and subsequently they were placed in media containing 1.71, 3.42, and 5.13 mM of Ca. The aim of this study was to investigate whether gamete fusion and the subsequent pronuclei formation resulting from the subzonal insemination is dependent upon the concentration of Ca in medium and to compare the effect of the Ca concentration on the fertilization of oocytes that had a sperm adhered to the oolemma and oocytes in which the sperm did not attach at the time of microinsemination.…”