Effect of Partial Incision of the Zona Pellucida by Piezo-Micromanipulator for In Vitro Fertilization Using Frozen-Thawed Mouse Spermatozoa on the Developmental Rate of Embryos Transferred at the 2-Cell Stage

Abstract

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.

Marked Improvement of Fertility of Cryopreserved C57BL/6J Mouse Sperm by Depletion of Ca2+ in Medium

“…Frozen C57BL/6J sperm exhibit acrosome damage with loss of contents caused by the freeze-thaw procedure [17], and almost no sperm can penetrate the zona pellucida. When partial zona pellucida nicking [18,19] or intracytoplasmic sperm injection (ICSI) [20,21] is performed to increase fertility, post-thawed C57BL/6J sperm can fertilize a high percentage of eggs. Improved IVF rates with intact oocytes have rarely been reported using motile sperm separated from thawed suspension [16,22] or methyl-beta-cyclodextrin (MBCD) stimulating cholesterol efflux from thawed sperm [23].…”

Effect of Zona Incision by Piezo-Micromanipulator (ZIP) on In Vitro Fertilization in 21 Transgenic Mice Lines

“…However, the fertility of cryopreserved spermatozoa from some inbred strains such as the C57BL/6 which is strain commonly used in transgenic and mutagenesis studies is extremely poor [4,10,15]. There are reports that methods of zona-pellucida dissection, partial zona-pellucida dissection (PZD) [9], partial zona-pellucida incision by piezo-micromanipulator (ZIP) [5], and laser-assisted zona drilling [3] are effective in increasing fertility rates. Especially, ZIP in combination with in vitro fertilization (IVF) using cryopreserved C57BL/6J transgenic spermatozoa dramatically improved fertilization rates and subsequent embryonic development [5].…”

“…There are reports that methods of zona-pellucida dissection, partial zona-pellucida dissection (PZD) [9], partial zona-pellucida incision by piezo-micromanipulator (ZIP) [5], and laser-assisted zona drilling [3] are effective in increasing fertility rates. Especially, ZIP in combination with in vitro fertilization (IVF) using cryopreserved C57BL/6J transgenic spermatozoa dramatically improved fertilization rates and subsequent embryonic development [5]. IVF with ZIP (ZIP/IVF) is also an effective assisted reproductive technique (ART) for reproduction of infertile transgenic mice with low-motility spermatozoa [6].…”

“…Approximately 2 µl of thawed sperm suspension was added to 400 µl of TYH medium. After incubation of the frozenthawed spermatozoa for 30 min at 37°C (for ZIP/IVF [5] and conventional IVF [9]) under 5% CO 2 in air, the ZIP oocytes and freshly collected oocytes with cumulus cells were separately introduced into TYH medium containing frozen-thawed spermatozoa. Fertilization was defined by the number of embryos that had developed to the 2-cell stage 24 h after insemination.…”

“…The ZIP procedure was essentially the same as described previously [5]. The zona pellucida was incised by the micropipette through the application of piezo pulses (controller setting: speed 2, intensity 2) while the pipette was moved along the surface of zona pellucida.…”