Nuclear Transfer of Synchronized African Wild Cat Somatic Cells into Enucleated Domestic Cat Oocytes

• Published in: Biology of reproduction Vol. 69; no. 3; pp. 1032 - 1041
• Main Authors: GOMEZ, Martha C, JENKINS, Jill A, GIRALDO, Angelica, HARRIS, Rebecca F, KING, Amy, DRESSER, Betsy L, POPE, Charles Earle
• Format: Journal Article
• Language: English
• Published: Madison, WI Society for the Study of Reproduction 01-09-2003
• Subjects: Animals; Biological and medical sciences; Blastocyst - physiology; Calcium - physiology; Carnivora - embryology; Cell Cycle; Cell Differentiation - physiology; Cell Division; Cells, Cultured; Cloning, Organism; Embryo Transfer; Embryology: invertebrates and vertebrates. Teratology; Embryonic and Fetal Development - physiology; Female; Fibroblasts - cytology; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Male; Molecular embryology; Nuclear Transfer Techniques; Oocytes - cytology; Oocytes - physiology; Pregnancy; Program Evaluation; Fissipedia; Embryonic development; Carnivora; Genetic variability; Endangered species; Cell nucleus; Wild animal; Synchronization; In vitro; Felis catus; Felis sylvestris; Vertebrata; Mammalia; Animal; Dedifferentiation; Domestic animal; Cell cycle; Transfer
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod.102.014449

The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear
transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction.
The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate
within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions
of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and
disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions
of AWC nuclei in the G 0 /G 1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in
the G 0 /G 1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin.
In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of
AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics
of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were
in the G 0 /G 1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition
(61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell
synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned
embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage
of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or
contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of
AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively.
The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher
percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei,
24.2% vs. 3.3%, respectively ( P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of
AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of
DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence
of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos.
In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6
or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were
observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high
rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint
to the in vivo developmental potential of the embryos.