Serum-starved apoptotic fibroblasts reduce blastocyst production but enable development to term after SCNT in cattle.
Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.
Miranda
Moysés dos Santos
Mdos S
Departamento de Ciências Básicas, FZEA-USP, Pirassununga-SP, Brazil.
Bressan
Fabiana Fernandes
FF
Zecchin
Karina Gottardello
KG
Vercesi
Anibal Eugênio
AE
Mesquita
Ligia Garcia
LG
Merighe
Giovana Krempel Fonseca
GK
King
William Allan
WA
Ohashi
Otávio Mitio
OM
Pimentel
José Rodrigo Valim
JR
Perecin
Felipe
F
Meirelles
Flávio Vieira
FV
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001755:Blastocyst / Q000166:cytology*; D002417:Cattle; D002453:Cell Cycle; D002467:Cell Nucleus / Q000235:genetics; D049109:Cell Proliferation; D065150:Cellular Reprogramming; D019976:Cloning, Organism / Q000379:methods*; D016895:Culture Media, Serum-Free; D047108:Embryonic Development; D005260:Female; D047109:Fetal Development; D005347:Fibroblasts / Q000473:pathology*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D010312:Parthenogenesis; D011247:Pregnancy; D018873:Pregnancy Rate
D016895:Culture Media, Serum-Free