28 PREGNANCIES RESULTED FROM GOAT NT EMBRYOS PRODUCED BY FUSING COUPLETS IN THE PRESENCE OF LECTIN

Abstract

The procedure of nuclear transfer (NT) using somatic cells remains inefficient partly due to low fusion rates between donor cells and recipient ooplasm. Lectin is a glycoprotein which specifically binds to carbohydrates to induce a tight contact of membrane to membrane (Booth et al., 2001 Cloning and Stem Cells, 3, 139–159). The purpose of this study was to examine the fusion rates and developmental competence of NT embryos following pre-incubation of couplets in medium containing lectin prior to electrical pulsing. Oocytes were collected by laparoscopic ovum pick-up from hormonally primed goats or by aspiration from culled goat ovaries, and cultured for maturation at 38.5°C, 5% CO₂. At approximately 24 h after the onset of IVM, the cumulus cells were stripped off by brief vortexing in medium containing 0.2% hyaluronidase. Oocytes with first polar bodies were selected for NT. Successful enucleation was confirmed by the absence of MII chromosomes in ooplasm by means of brief exposure of the Hoechst 33342-stained oocytes to UV light. Three cumulus-granulosa cell lines from transgenic goats were used as donor cells. They were cultured to confluency in DMEM + 20% FCS for 6 days prior to NT. Individual donor cells were transferred into the perivitelline space of the enucleated oocytes. Couplets were incubated for 15 minutes in TCM199 + 10% FCS containing 75 or 150 μg mL⁻¹ lectin (L-9132 Sigma, St.Louis, MO, USA) prior to being subjected to electrical pulsing (lectin treatment) with one DC pulse at 2.4 kV/cm (1st pulsing). The fusion rate was determined 40–60 minutes after the 1st electric pulsing. Non-fused couplets were exposed to a 2nd pulsing. Approximately 30 minutes later, non-fused couplets were exposed to a 3rd pulsing. Couplets without the lectin treatment served as controls. Reconstructed embryos were activated with 5 μM ionomycin followed by 5 h of incubation in 10 μg mL⁻¹ cycloheximide with 7.5 μg mL⁻¹ cytochalasin B. A group of 10 to 13 embryos was transferred into a recipient after 12 to 14 h of culture in G1.3. Statistical analysis was performed using the χ² analysis. Results are shown in the following table. This study demonstrated that fusion rate could be improved by pre-incubating couplets in the medium containing 150 μg mL⁻¹ lectin prior to electrical pulsing and the embryos derived from the lectin treatment could establish the early pregnancies.