K. Villemoes

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.

High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification.

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4+/-2.4%) or 130 (13.1+/-3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 degrees C or 25 degrees C. Oocytes pressurized at 37 degrees C had a significantly higher blastocyst (14.1+/-1.4%) rate than those treated at 25 degrees C (5.3+/-1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.

Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos.

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41-42 h oocytes maturation, the oocytes were further cultured with or without 0.4 microg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 +/- 2% vs 48.1 +/- 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.

Developmental potential and kinetics of pig embryos with different cytoplasmic volume.

The effects of cytoplasmic volumes on development and developmental kinetics of in vitro produced porcine embryos were investigated. During hand-made cloning (HMC), selected cytoplasts were separated into two groups according to their size in relation to the initial oocyte: ~75% or ~50%. Following two fusion steps and activation (day 0), reconstructed embryos were cultured in vitro for 6 days. Cleavage rates on day 2 as well as blastocyst rates and cell numbers on day 6 were recorded. Results showed that embryo development was no different for ~50% versus ~75% cytoplasm at first fusion. This result was used in the following experiments, where the effect of varying cytoplasm volume in second fusion to obtain a final cytoplasm volume of ~75% to ~200% was tested. The results showed that the lowest quality was obtained when the final cytoplasm volume was ~75% and the highest quality at ~200% of the original oocyte. Similar results were observed in parthenogenetic (PA) embryos activated with different cytoplasmic volumes. A common pattern for the developmental kinetics of HMC and PA embryos was observed: the smaller group tended to have a longer time for the first two cell cycles, but subsequently a shorter time to form morula and blastocyst. In conclusion, the developmental kinetics of in vitro produced embryos was affected by the cytoplasm volume of the initial oocyte, and this further accounted for the developmental ability of the reconstructed embryos.