Fertilization and Development of Mouse Oocytes Injected with Isolated Sperm Heads1

Abstract

To determine whether spermatozoa must be structurally intact before microsurgical injection into oocytes for normal fertilization, intact spermatozoa, as well as sperm heads separated from tails by sonication, were individually injected into oocytes. When whole spermatozoa were injected immediately after their immobilization, the majority of the oocytes were fertilized and developed normally. Sonication in the presence or absence of Triton X-100 decapitated more than 95% of spermatozoa. Although all decapitated spermatozoa were diagnosed as "dead" by live/dead sperm staining, separated sperm heads (nuclei) could participate in normal embryo development when injected into the oocytes. The ability of isolated sperm heads (nuclei) to participate in normal embryo development was maintained under cryopreservation conditions that were not suitable for the survival of plasma membrane-intact spermatozoa. These results indicate that 1) spermatozoa do not need to be structurally intact for intracytoplasmic injection, 2) the plasma and acrosomal membranes and all tail components are not essential for normal embryo development, at least in the mouse, and 3) the cryopreservation conditions required for maintenance of the genetic integrity of sperm nuclei are less stringent than those necessary for keeping plasma membrane-intact spermatozoa alive.

Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring

“…One thing to be stressed here is that the type of medium used for suspension of defrosted spermatozoa and spermatids makes a difference in the outcome of the experiments: the birth of normal offspring. Potassium-rich Ca 2ϩ -and Mg 2ϩ -free NIM medium (13,19) always gave better results (Tables 2 and 3). Ca 2ϩ -containing ordinary cell culture media like PBS may activate endogenous nucleases (20), which attack the DNAs of these spermatozoa and spermatids with broken plasma membranes.…”

“…There was a significant effect of cell type on both implantation and birth rates (P Ͻ 0.05; one-way ANOVA). Results, summarized in Table 2, indicated that spermatozoa collected from frozen cauda epididymis produced live offspring when defrosted spermatozoa were suspended in K ϩ -rich nucleus isolation medium (NIM) (13), not conventional GL-PBS medium (Dulbecco's PBS supplemented with 5.6 mM glucose͞5.4 mM sodium lactate͞5 mg͞ml BSA͞0.01% polyvinyl alcohol; ref. 12), before injection into oocytes (P Ͻ 0.05; vs. suspension in GL-PBS).…”

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

“…The isolated sperm head was injected immediately into an oocyte. Only the sperm head is needed to obtain offspring by ICSI (15). A single human, bull, or boar spermatozoon, drawn into the pipette, was immobilized by applying a few piezo pulses to its midpiece region before the entire body of the spermatozoon was injected into a mouse oocyte.…”

Improvement of mouse embryo quality by myo-inositol supplementation of IVF media

“…ICSI was carried out by the procedure described [26,28], under a Nikon (Nikon Corp., Japan) invertoscope equipped with Leitz (Wild Leica, Wetzlar, Germany) mechanical micromanipulators, Märzhäuser (Wetzlar, Germany) piezoelectric apparatus and using Humagen (Charlottesville, VA, USA) holding and ICSI micropipettes. A 1 μL aliquot of sperm suspension was mixed thoroughly with a droplet of 50 μL Quinn's Sperm Washing Medium containing 10 % (w/v) polyvinylpyrrolidone (Mr 360,000).…”