Beneficial effect of young oocytes for rabbit somatic cell nuclear transfer

Abstract

This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p < 0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R = 0.326; blastocyst rate vs. MAPK, R = -0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n = 117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12 h after hCG injection, was beneficial for the development of NT embryos.

FIG. 1.

Rabbit oocyte recovery at different times post-hCG injection. MII oocytes collected from Graafian follicles were defined as follicular oocytes, and those collected from flushed oviducts, as oviductal oocytes. Both follicular and oviductal oocytes were collected at 10 (n = 194), 12 (n = 921), 14 (n = 454), and 16 (n = 733) hpi. These values were calculated and classified into three categories: follicular, oviductal, and total average number of oocytes per donor.

FIG. 2.

Analysis of MPF and MAPK activity in oocytes of different ages. (A) A representative autoradiograph of histone H1 and myelin basic protein (MBP) kinases in the oocytes collected at 10 (n = 58), 12 (n = 41), 14 (n = 35), and 16 (n = 80) hpi. (B) Relative activity levels of MPF (shown as H1 activity) and MAPK (shown as MBP activity) for oocytes collected at 12, 14, and 16 hpi were compared to the standardized values of those harvested at 10 hpi.

FIG. 3.

Cloned rabbits generated by nuclear transfer (NT) from adult somatic cells. (A) Enucleation of matured rabbit oocyte by pressing out the first polar body (indicated by arrow) along with a portion of cytoplasm through an incision in zona pellucida made by a microneedle. The metaphase plate and polar body chromosomes (near arrow heads) were visualized in the extruded cytoplasm by fluorescent microscopy after staining with 10 μg/mL Hoechst 33342 dye (top right window). Only enucleated oocytes, whose MII chromosomes were confirmed to be contained in the cytoplasm removed, were used for NT. (B) During NT, partially lysed cumulus cells containing nucleus (arrow) ready to inject into cytoplasm of enucleated oocyte by direct nuclear injection, or (C) an intact cumulus cell transferred into the perivitelline space of an enucleated oocyte (arrow) prior to membrane fusion. (D) Cloned rabbit embryos developed to four-celled stage 16 h after culture in vitro, and (E) continued development into hatched blastocyst stage after 5 days of culture, containing 596 stained nuclei (F). Pregnancy was determined by ultrasound on day 14 after embryo transfer (ET); (G) fetus body indicated by arrows, and yolk sac cavity by arrowhead. (H) The first full-term rabbit clone (Clone A) was naturally delivered as a stillborn 33 d after ET, and (I) the second clone (arrow, Clone B) was delivered alive, by C-section, 31 days after ET. Bar = 100 μm.