Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development

Abstract

In mice and humans, a normal offspring can be obtained by injecting a single spermatozoon into an oocyte, the process called intracytoplasmic sperm injection (ICSI). When three or more mouse spermatozoa with intact acrosomes were injected into individual mouse oocytes, an increasing proportion of oocytes became deformed and lysed. Oocytes did not deform and lyse when acrosome-less spermatozoa were injected, regardless of the number of spermatozoa injected. Injection of more than four human spermatozoa into a mouse oocyte produced vacuole-like structures in each oocyte. This vacuolation did not happen when spermatozoa were freed from acrosomes before injection. Hamsters, cattle, and pigs have much larger acrosomes than the mouse or human. Injection of a single acrosome-intact hamster, bovine, and porcine spermatozoon deformed and lysed many or all mouse oocytes. This deformation did not happen when these spermatozoa were freed from acrosomes before ICSI, regardless of the number of spermatozoa injected. Because trypsin and hyaluronidase mimicked the action of acrosome-intact spermatozoa, it is likely that the acrosomal enzymes deform and lyse the oocytes. Injection of small amounts of trypsin and hyaluronidase into normally fertilized mouse eggs disturbed their pre- and postimplantation development. In view of potentially harmful effects of acrosomal enzymes on embryo development, the removal of acrosomes before ICSI is recommended for animals with large sperm acrosomes. The removal of acrosomes may increase the efficiency of ICSI in these animals. Although human and mouse spermatozoa do not need to be freed from acrosomes, the removal of acrosomes before ICSI is theoretically preferable.

Fig. 1.

Mouse oocytes each injected with a single or multiple mouse sperm heads. (A) A deformed oocyte 7 h after injection of four acrosome-intact sperm heads. (B) An oocyte cytolyzed after injection of 10 acrosome-intact sperm heads. (C) An oocyte injected with 4 acrosome-less sperm heads. This oocyte did not deform and contained a few large pronuclei. (D) Confocal image of an oocyte 7 h after normal IVF. Note many asterous tubules in the ooplasm. Pronuclei are shown as large red spherical structures. (E) Confocal image of an oocyte injected with six acrosome-intact sperm heads. Note that microtubules are lying near the cortex of the deformed oocyte and are absent in the interior part of the oocyte. Pronuclei are not in this optical section. [Scale bars: 10 μm (A–C), 10 μm (D), and 10 μm (E).]

Fig. 2.

Mouse oocytes injected with hamster or human spermatozoa. (A)An oocyte injected with a single acrosome-intact hamster sperm head, 4 h after ICSI, with extensive deformation. (B) An oocyte injected with a single acrosome-intact hamster sperm head, 24 h after ICSI, with extensive deformation. (C) An oocyte injected with a single acrosome-less hamster sperm head, 4 h after ICSI, without deformation. (D) An oocyte injected with a single acrosome-intact human spermatozoon, 6 h after ICSI, without deformation. (E)An oocyte injected with six acrosome-intact human spermatozoa, 20 h after ICSI, with irregular appearance of the cytoplasm. (F) Confocal section of an oocyte injected with six acrosome-intact human spermatozoa, 20 h after ICSI, showing many “vacuoles” within the cytoplasm. Sperm pronuclei are shown as red spherical structures. (A–E) Interference-contrast microscopy. (F) Confocal scanning laser microscopy. [Scale bars: 10 μm (A–E) and 10 μm (F).]