ABSTRACT
The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at -20 degrees C until use. When the sperm heads were aspirated into injection pipettes 7-10 microm in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2-4 microm in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague-Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.
