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EFFECT OF PARTIAL ZONA-PELLUCIDA INCISION BY PIEZO-MICROMANIPULATOR ON THE FERTILITY OF FROZEN-THAWED MOUSE SPERMATOZOA AND SUBSEQUENT EMBRYO TRANSFER.
Kawase, Yosuke1, Iwata, Takamitsu1, Ueda, Otoya1, Tachibe, Takanori1, Aoki, Yukari1, Kaneko, Takehito2, Nakagata, Naomi2, Suzuki, Hiroshi1, 1 2
ABSTRACT- Cryopreservation of mouse spermatozoa has become available for use. Most transgenic mouse lines are established by DNA microinjection into C57BL/6 zygotes, but unfortunately, the fertilizing ability of crypreserved C57BL/6 spermatozoa is very low compared with other inbred strains. We have recently solved this difficulty by in vitro fertilization in combination with partial zona-pellucida dissection (PZD). However, there is a limitation that fertilized eggs with PZD require to culture in vitro up to morula or blastocyst stage before embryo transfer. On earlier stage of embryos, blastomeres escape from the slit of zona-pellucida and attach to epithelial cells of oviduct. To overcome this problem, we applied partial zona-pellucida incision by piezo-micromanipulator, which has been used for intracytoplasmic sperm injection in mice, for the in vitro fertilization using frozen-thawed mouse spermatozoa. In preliminary experiment, the blunt-ended tip of micro-pipette was touched with the surface of zona-pellucida of fertilized eggs at pronucleus stage. The zona-pellucida was incised by the micro-pipette with applying piezo pulses while the pipette was moved along by the surface of zona-pellucida. The length of the zona-pellucida incision by Piezo-maicromanipulator (ZIP) was 2r/12 um or 4r/12 um. As a control, the zona-pellucida of fertilized eggs was partially dissected by the method of Nakagata et al. 46% of transferred eggs with ZIP 2r/12 um in length into the oviduct of recipient mice developed to term. When PZD eggs were transferred into the oviduct, only 9% of the eggs developed to newborn. None of transferred eggs with ZIP 4r/12 um in length developed to term. In the next series experiment, frozen-thawed genetically modified C57BL/6J spermatozoa were inseminated with ZIP 2r/12 um in length and PZD oocytes. Fertilization rates of those oocytes were 52% and 48%, respectively. When those fertilized eggs at 2-cell stage were transferred into the oviduct, 18% and 2% of the transferred embryos with ZIP and PZD were developed to term, respectively. This difference was statistically significant (P<0.05). These results indicate that ZIP is an effective ART for in vitro fertilization using cryopreserved mouse spermatozoa and subsequent embryo transfer.
KEY WORDS: mouse, cryopreservation, spermatozoa, ART
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