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  • 1
    Electronic Resource
    Electronic Resource
    Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 31 (1992), S. 223-229 
    Details
    ISSN: 1040-452X
    Keywords: Mouse ; Spermatozoa ; Acrosome reaction ; Sperm injection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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〈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 off-spring (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.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080310310
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  • 2
    Electronic Resource
    Electronic Resource
    Calcium concentration and fertilization by subzonal insemination of a single spermatozoon in mouse oocytes (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 54-60 
    Details
    ISSN: 1040-452X
    Keywords: Mouse spermatozoa ; Subzonal insemination ; Calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of calcium concentration in culture medium on the fertilization of subzonally microinseminated mouse oocytes was examined. Oocytes were injected with a single spermatozoon so that the sperm head was forced to adhere onto the ooplasmic membrane with a micromanipulation technique. For the inseminations, epididymal spermatozoa preincubated in culture medium and those treated with ionophore A23187 were used. Inseminated oocytes were cultured using media with three different calcium concentrations of 1.71, 3.42, and 5.13 mM; 40.0%, 71.6%, and 47.9% of oocytes microinjected with preincubated sperm were fertilized after incubation with those media, respectively. When the oocytes inseminated with ionophore-treated sperm were incubated in media containing 1.71 and 3.42 mM calcium, their fertilization rates were 58.2% and 87.5%. Thus fertility of subzonally microinseminated oocytes was obviously enhanced when cultured in medium with 3.42 mM of calcium, irrespective of being inseminated with preincubated sperm (P 〈 0.01) or with ionophore-treated sperm (P 〈 0.005). Some of the microinseminations with preincubated sperm were performed without sperm adhered to the oolemma. In these cases, the incidence of fertilization was not improved by incubating the inseminated oocytes in medium containing 3.42 mM calcium (32.6%) as compared to those incubated in medium with 1.71 mM calcium (28.3%). These results suggest that the concentration of extracellular calcium exerts an important effect on the progress of fertilization events subsequent to sperm adherence onto the ooplasmic membrane. Almost 80% of the zygotes fertilized via incubation in medium with 3.42 mM of calcium developed into blastocysts after culturing in vitro. © 1994 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080380110
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  • 3
    Electronic Resource
    Electronic Resource
    Subzonal insemination of a single mouse spermatozoon with a personal computer-controlled micromanipulation system (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 33 (1992), S. 81-88 
    Details
    ISSN: 1040-452X
    Keywords: Sperm injection ; lonophore treatement ; Computer-controlled microsurgery ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A personal computer-controlled micromanipulation system was developed for automatic injection of spermatozoa into the perivitelline space of mouse ova. A pair of three-dimensional hydraulic micromanipulators driven by pulse motors was used for this automatic system. The pulse signals that regulate the motors are initiated by the computer program, and these signals cause the micromanipulator to move the microtool precisely. The computer program was designed to perform the most effective movements of the sperm injection needle used during manual micromanipulation. Prior to the manipulation, the computer locates the tip of the injection needle and the end of the egg-holding pipette in the microscope field using image processing. The trajectory of the injection needle is determined according to these initial positions. Using this robotic system, subzonal insemination with a single mouse spermatozoon was attempted in a total of 143 ova. The sperm insertion was successfully completed in all cases without damaging any of the ova. Spermatozoa treated with ionophore A23187 and those without the treatment were used. The fertilization rate (68.8%) of the ova inseminated with treated sperm was significantly higher than that (37.5%) obtained with the nontreated sperm (P 〈 0.05). These findings suggest the feasibility and potential for further applications of a robotic microinsemination system and, in addition, that a higher fertility rate in the subzonal insemination of mouse ova can be achieved with the ionophore treatment of spermatozoa. © 1992 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080330112
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