Fertilization through spermatozoal microinjection: significance of acrosome reaction

The acrosome reaction in mouse spermatozoa was induced by various means. These were 1) varying incubation time in T6 medium, 2) incubation in T6 medium with added A23187, 3) incubation in T6 medium with added dbcGMP and imidazole, 4) exposure to an electric field, and 5) a combination of incubation in a medium with dbcGMP and imidazole and electroporation. The mean percentages of acrosome-free spermatozoa obtained by these various methods and assessed on the basis of both Bryan's stain and immunolocalization by FITC-labeled monoclonal antibodies increased by steps from 36% to 67%, 73%, 86%, and 92%. Individual spermatozoa from the various treatments were afterwards microinjected under the zona pellucida of a mouse oocyte. The fertilization rate for eggs microinjected with a spermatozoon treated with A23187, dbcGMP, and imidazole, by electroporation and by a combination of the last two methods also increased by steps from 17% to 34%, 36%, and 70%, respectively. Ninety-five percent of the fertilized oocytes reached the early blastocyst stage, thirty-eight percent of these blastocysts implanted in pseudopregnant mice, and twenty-eight percent developed to term. These results indicated the varying degrees of success of different ways of inducing acrosomal loss in spermatozoa and their subsequent success rates in fertilization and further in vitro and in vivo development.

Section: Discussionsupporting

confidence: 69%

Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs

Palermo¹,

Steirteghem²

1991

“…Our results are in contrast to those obtained by Yamada et al (1988) and Mann (1988). Yamada et al (1988) reported on diploid mouse embryos after sperm microinjections, but they gave no evidence that the sperm genome was included in the chromosomal complement of the embryo. However, Mann (1988) reported on newborn mice from microinjected oocytes.…”

Section: Discussioncontrasting

confidence: 99%

“…In nearly all experiments performed until now, the microinjection of sperm under the zona pellucida failed to result in fertilization (Westhusin, 1986;Barg et al, 1986;Green, 1987) or embryonic development (Metka et al, 1985;Laws-King et al, 1987;Lassalle et al, 1987;Yamada et al, 1988). Mostly, fertilization was proven by the presence of a second polar body and two pronuclei.…”

Section: Introductionmentioning

confidence: 99%

Subzonal microinjection of mouse spermatozoa: Insufficient sperm motility might induce phagocytosis

Klemm

Engel

1991

Acrosome-reacted CB6F1 mouse spermatozoa with slight flagellar motility were microinjected under the zona pellucida of CB6F1 mouse oocytes. Electron microscopy revealed the presence of swollen and decondensed sperm heads in the oocyte cytoplasm. Sixty-one percent of the microinjected oocytes reached a morphologically apparent two-cell stage, but chromosomal analysis demonstrated only haploid chromosomal complements in all cases. The exposure of microinjected oocytes to suspensions of spermatozoa of mice homozygous for a 2,4 reciprocal translocation resulted in normal fertilization and embryonic development with a maternally as well as a paternally derived haploid genome. Identical results were obtained with oocytes microinjected with medium and subjected to in vitro fertilization thereafter. Thus it can be suggested that the microinjected spermatozoa with insufficient flagellar motility are incorporated into the oocyte cytoplasm by phagocytosis. These spermatozoa do not induce a polyspermy block but induce the oocyte to parthenogenetic development.

“…For these reasons, the sperm injection needle always passes through the ZP successfully, even when the operator is inexperienced. Yamada et al (1988) presented a procedure for the insertion of the sperm injection needle into the PVS of mouse oocytes with a series of photographs. According to their method, the path of the sperm injection needle through the ZP differs from that of the force used to hold the ovum.…”

Section: Procedures For Microinjection Of Sperm Intomentioning

confidence: 99%

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

Kobayashi

Okuyama

Fujimoto

et al. 1992

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.