66 EFFECTS OF CONTACT INHIBITION INTERVALS V. SERUM DEPRIVATION ON DEVELOPMENT OF PORCINE NUCLEAR TRANSFER DERIVED EMBRYOS

Abstract

Contact inhibition and serum deprivation are commonly used to synchronize donor cells at the G0/G1 state of the cell cycle prior to use in nuclear transfer. Here we compared the effects of serum deprivation (SD) and different intervals of contact inhibition (CI) of the donor cells on the blastocyst rate. One batch from pooled porcine fetal fibroblasts (passage 3) was used in this study. The cells were thawed, seeded to a six-well plate and cultured in Dulbecco‘s Modified Eagles Medium (DMEM) supplemented with 2 mM glutamine, 1% non-essential amino acids, 0.1 mM mercaptoethanol, 100 U mL⁻¹ penicillin, 100 mg mL⁻¹ streptomycin containing 10% fetal calf serum (FCS). Serum deprivation was achieved by culturing cells in DMEM containing 0.5% FCS for 48 h. Cells of the CI groups were grown to 100% confluency and kept in that state for 24 h, 48 h and 72 h. Immediately after cell cycle synchronization, cells were used in nuclear transfer. Cell cycle state of the cells was evaluated by FACS analysis at 24 h after beginning of CI and prior to nuclear transfer. Blastocyst rate was determined 7 days after nuclear transfer. An average of 38-42 h in vitro matured oocytes were used in nuclear transfer (NT). NT was performed as described previously (Betthauser J et al., 2000 Nat. Biotechnol. 17, 456–461). There were no differences in the proportion of cells in G0/G1 of the cell cycle in any of the treatment groups (85.0%, 85.8%, 85.5% and 86.3% for SD and CI at either 24 h, 48 h and 72 h, respectively). After nuclear transfer (for each CI group n = 336–384 reconstr. embryos; SD n = 215) there was a statistically significant difference in the fusion rate between 48 h CI and SD cells (74.8% v. 87.5%, t-test P < 0.050). Blastocyst rate (blastocysts/fused) differed significantly between SD, 24 h CI and 48 h CI (17.4%; 9.1%, 9.6%, t-test P < 0.050), there was no difference between SD and 72 h CI and within the CI groups (72 h CI 10.6%). Four transfers of reconstructed embryos (72 h CI, n = 138–163 embryos/gilt, 1-cell embryos) to prepuberal Landrace gilts led to 2 initial pregnancies determined at Day 25 by ultrasound. One pregnancy was lost at Day 35; the other recipient remained pregnant and farrowed 4 piglets. One piglet was stillborn and one died 7 h after birth; the remaining two piglets are healthy and now 4 months old. Four transfers of embryos (n = 96–110) reconstructed with SD cells revealed two initial pregnancies determined on Day 25 by ultrasound. Again, one was lost on Day 35, and the other one is now at Day 100. Our results show that, despite similar proportions of cells being in G0/G1 of the cell cycle, cells either contact-inhibited for 72 h or serum-deprived both show higher rates of blastocyst development compared to cells contact-inhibited for shorter time periods. Both donor cell preparations can lead to full term development of nuclear transfer-derived embryos. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SFB 265).