Germinal Vesicle Transfer Between Fresh and Cryopreserved Immature Mouse Oocytes

Abstract

Objective: Germinal vesicle (GV) transfer offers the potential to assess and rescue genomic maturation in mammalian oocytes. However, as a clinical or research procedure, this technique presents a major logistic problem to ensure that germinal vesicle oocytes are obtained in the same cell cycle stage. This problem could be eliminated if cryostored, immature oocytes are shown to be appropriate for use as GV donors in the transfer procedure. Design: Genomic maturation, as judged by first polar body extrusion, was assessed in mouse oocytes reconstructed by GV transfer using nuclear and/or cytoplasmic components from cryopreserved oocytes. Materials and Methods: Female mice (CD-1 strain) were treated with PMSG (10 IU ip). The mice were killed at 36–48 h post injection and the eggs harvested by follicular puncture. Oocytes were frozen in propanediol-sucrose using routine slow embryo freezing and rapid thawing protocols. GV transfer was performed to exchange nuclei between fresh and frozen oocytes; the transfer and electrofusion protocols have been described previously (Liu et al, Human Reproduction 14:2357, 1999). Throughout these procedures, the oocytes were treated with isobutyl-methylxanthine to block meiotic progression. Following electrofusion the eggs were cultured in HTF containing 10% fetal bovine serum and monitored between 16–20 h for first polar body extrusion. Staining with Hoechst 3342 revealed DNA in both oocyte and polar body. Results: legendPreparationlegendThe survival rate for the freeze-thaw procedures was 76% (679/890).Electrofusion rateMaturation rateFresh GV-fresh cytoplasm41% (55/135)80% (44/55)Frozen GV-frozen cytoplasm41% (57/138)91% (52/57)Fresh GV-frozen cytoplasm51% (73/143)86% (63/73)Frozen GV-fresh cytoplasm40% (49/124)78% (38/49)Fresh control (no micromanipulation)–77% (167/218)Fresh control (electrofusion pulses only)–86% (36/42)Frozen control (no micromanipulation)–79% (216/275)legend The survival rate for the freeze-thaw procedures was 76% (679/890). Open table in a new tab Conclusions: Immature oocytes reconstructed by GV transfer with nuclei or cytoplasm from frozen eggs show comparable electrofusion and maturation rates as non-manipulated fresh oocytes. These results suggest cryobanked immature oocytes may be suitable for use in research or clinical GV transfer procedures.