Intracytoplasmic sperm injection (ICSI) was successful in the mouse when a piezo-driven micropipette was used instead of a mechanically driven conventional pipette. Eighty percent of sperm-injected oocytes survived, and approximately 70% of them developed into blastocysts in vitro. When 106 embryos at the 2- to 4-cell stage were transferred to eight naturally mated foster mothers, 30% of the embryos (25-43%, depending on the host) reached the full term. Except for two that were cannibalized soon after birth, all of the young (30 pups) grew into normal adults. Studies of this type on the mouse may increase understanding of the fertilization process and of how ICSI works.

Fiber-shaped H-aggregates with lengths of up to 300 microm are synthesized by self-assembly of thiacyanine (TC) dye molecules in solution. Photoluminescence (PL) images and spatially resolved PL spectra of the fibers that are transferred onto a glass substrate reveal that the fibers act as single-mode optical waveguides that propagate PL in the range of 520 to 560 nm over 250 microm without any loss.

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.

The mouse oocyte can be activated by injection of a single, intact mouse spermatozoon or its isolated head. Isolated tails are unable to activate the oocyte. Active sperm-borne oocyte-activating factor(s) (SOAF) appears during transformation of the round spermatid into the spermatozoon. The action of SOAF is not highly species-specific: mouse oocytes are activated by injection of spermatozoa from foreign species, such as the hamster, rabbit, pig, human, and even fish. Some SOAF can be extracted by simple freeze-thawing of (hamster) spermatozoa; additional SOAF is obtained by sequential treatment of spermatozoa with Triton X-100 and SDS. Electron microscopic examination of sperm heads during SOAF extraction suggests that the relatively insoluble SOAF is associated with perinuclear material. When microsurgically injected into oocytes, Triton X-100-treated sperm heads (with perinuclear material, but without any membranes) can activate the oocytes, leading to normal embryonic development. Whereas perinuclear components have been believed to play a purely structural role, these data suggest an additional function for them in oocyte activation.

This study shows that the nucleus of the secondary spermatocyte can participate in syngamy and normal embryonic development. Spermatogenic cells were released from the seminiferous tubules of adult mice, and the secondary spermatocytes were selected according to the size of the whole cell and nucleus. The accuracy of this selection, evaluated by chromosome analysis, was 86%. Nuclei of presumptive secondary spermatocytes were freed from the cytoplasm and then injected individually into mature oocytes. This process itself did not activate the oocytes. The oocytes were electroactivated about 2 h after injection, at which time prematurely condensed chromosomes of the spermatocyte had become associated with the microtubules of a spindle. Following activation, the chromosomes of both the oocyte and spermatocyte completed their second meiotic division, culminating in the extrusion of two separate polar bodies and the formation of one male and one female pronucleus in about 75% of the oocytes into which spermatocyte nuclei had been injected. Two- or four-cell embryos arising from such oocytes were randomly selected and transplanted to foster mothers. Twenty-four percent developed into normal fertile offspring. The young born later to these offspring were all normal. The results of this study indicate that gametic imprinting of mouse spermatogenic cells is completed either in the testis before the second meiotic division or within the cytoplasm of a mature oocyte after artificial nuclear transfer.

Although sonication is a simple way to immobilize ("kill") spermatozoa prior to injection into oocytes, this has been thought to be destructive to sperm chromosomes. Mouse and human spermatozoa were immobilized by sonication and kept in various media for up to 2 h, then their nuclei were individually injected into mouse oocytes for the analysis of chromosomes at the first cleavage metaphase. In both the mouse and human, incidence of structural chromosome aberrations was much higher in the spermatozoa sonicated and stored in Biggers-Whitten-Whittingham medium for 2 h at 37.5 degrees C than in those stored for 5 min in the same medium. We concluded, therefore, that it is not sonication per se but a prolonged exposure of sperm nuclei to extracellular milieu that is detrimental to sperm chromosomes. The incidence of structural chromosome aberrations of mouse and human spermatozoa was significantly reduced when the spermatozoa were sonicated and stored in K(+)-rich nucleus isolation medium containing EDTA. This suggests that sperm chromosome degradation following sperm immobilization by sonication is partly due to detrimental effects of a Na(+)-rich medium and of DNase on sperm chromatin. Ideally, it should be possible to prepare artificial media that maintain the integrity of sperm chromosomes for many hours after immobilization.

The self-propelled motions of micron-sized nematic liquid crystal droplets in an aqueous surfactant solution have been studied by tracking individual droplets over long time periods. Switching between self-propelled modes is observed as the droplet size decreases at a nearly constant dissolution rate: from random to helical and then straight motion. The velocity of the droplet decreases with its size for straight and helical motions but is independent of size for random motion. The switching between helical and straight motions is found to be governed by the self-propelled velocity, and is confirmed by experiments at various surfactant concentrations. The helical motion appears along with a shifting of a point defect from the self-propelled direction of the droplet. The critical velocity for this shift of the defect position is found to be related with the Ericksen number, which is defined by the ratio of the viscous and elastic stresses. In a thin cell whose thickness is smaller than that of the initial droplet size, the droplets show more complex trajectories, including "figure-8s" and zigzags. The appearance of those characteristic motions is attributed to autochemotaxis of the droplet.

Intracytoplasmic sperm injection (ICSI) has wide clinical application. In order to achieve good results with this method, it is important to restrict the possibility of oocyte injury as much as possible, and securely inject spermatozoa into the ooplasm. For this purpose, we clinically applied piezo-ICSI, which employs a micromanipulator with piezoelectric elements, to humans, and compared the results with those obtained by conventional ICSI. Conventional ICSI and piezo-ICSI were used in 279 cycles and 335 cycles respectively. Piezo-ICSI showed significantly more favourable results, with a survival rate of 88.1% (conventional ICSI: 81.4, P < 0.001), a fertilization rate of 79.4% (conventional ICSI: 66.4%, P < 0.001), and a pregnancy rate of 23.1% (conventional ICSI: 14.9%, P < 0.05). In piezo-ICSI, the needle used is not sharpened and has a flat tip. However, deformation of the oocyte during insertion of the needle is restrained by vibration of the piezo, and the oolemma is punctured readily and securely by the piezo pulse, at the site where the spermatozoon is injected. Piezo-ICSI is a promising new technique for human ICSI that should improve the survival, fertilization and pregnancy rates after ICSI.