1 DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS CLONED FROM FOLLICULAR DONOR CELLS IN SEQUENTIAL STAGES OF DIFFERENTIATION

Abstract

Efficient production of cloned embryos and live offspring is dependent on the ability of the nuclear-donor cell to be reprogrammed to direct normal conceptus development. Results of comparative studies indicate that embryonic and fetal cells are more successful candidates for nuclear transfer (NT) than terminally differentiated cells. Comparison of donor-cell efficiency is difficult to interpret across laboratories and from donor animals of varying genetic backgrounds and tissues of origin. This study was undertaken to determine the effect of the stage of differentiation of adult somatic donor cells derived from one-cell lineage of an individual donor animal. The follicular cell lineage including preantral follicle (PAFC), cumulus (CC), granulosa (GC), and luteal cells (LC) was chosen as several cell types in the lineage have previously proven successful for NT. Donor cell cultures were established from a 3-year-old Holstein cow. Embryos were reconstructed using confluent, early passage cultures. For each replicate, embryos were produced from two donor cell types in randomized block design (14 trials). Viable embryos were transferred to recipient females after 7 days of in vitro culture. Pregnancy and fetal viability were monitored weekly by ultrasonography from Days 30–100 of gestation and by rectal palpation thereafter. Embryos reconstructed from PAFC were less likely to develop to the blastocyst stage on Day 7 than embryos derived from CC or LC (Table 1, P < 0.05). Pregnancy rates at Day 30 were similar across donor cell types. A greater proportion of PAFC embryos were viable at Day 60 of gestation than embryos derived from CC and GC (P < 0.05). To date, normal cloned calves have been delivered at term from CC and GC, and two pregnancies (n = 3 fetuses) are ongoing from PAFC. The fetus cloned from LC, the terminally differentiated cell type in the lineage, was spontaneously aborted at day 211 with congenital abnormalities. Results from comparative studies of development of mouse embryos cloned from embryonic stem (ES) cells v. somatic donor cells indicate that ES-derived clones are less efficient in blastocyst formation, but survival to term is greater (Humpherys D et al., 2002 PNAS 99, 12 889–12 894). Likewise, our results in cattle suggest that PAFC, the least differentiated cells in the lineage, result in fewer cloned blastocysts, but blastocysts that do develop are more likely to progress through implantation and into later stages of pregnancy.

Table 1. 
Development of NT embryos reconstructed from follicular donor cells at sequential stages of differentiation