Isolation of apoptotic mouse fetal oocytes by AnnexinV assay.
Expression of phosphotidylserine by fetal oocytes in culture renders significant numbers of such cells able to bind AnnexinV-coated microbeads and allows their separation from Annexin V-negative oocytes on a Magnetic Cell Separation (MACS) column in a magnetic field. The majority of oocytes (> or =75%) which bound Annexin V-coated microbeads were viable, as indicated by their propidium iodine (PI) negativity. However, they showed apoptotic morphologies and were found to be TUNEL-positive. On the other hand, AnnexinV-negative oocytes, besides being PI negative, appeared morphologically healthy and TUNEL negative. Moreover, AnnexinV-positive oocytes showed a marked lower ratio of Bcl-xL/Bax transcripts in comparison to AnnexinV-negative oocytes. We conclude that the present method is able to separate fetal oocytes in two distinct populations: AnnexinV-positive oocytes showing features typical of apoptotic cells and AnnexinV-negative oocytes comprising for the most part viable non-apoptotic cells. This procedure should greatly facilitate studies aimed to identify the currently poorly understood molecular pathways governing apoptosis in mammalian fetal oocytes.
Lobascio
Anna-Maria
AM
Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00173 Rome, Italy.
Klinger
Francesca-Gioia
FG
De Felici
Massimo
M
D000818:Animals; D017304:Annexin A5 / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D005260:Female; D020287:In Situ Nick-End Labeling; D051379:Mice; D009865:Oocytes / Q000166:cytology* / Q000502:physiology*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D051028:bcl-2-Associated X Protein / Q000235:genetics; D051020:bcl-X Protein / Q000235:genetics
D017304:Annexin A5; D015415:Biomarkers; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein