Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

Abstract

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

Fig 1. Delayed initiation of germinal vesicle breakdown (GVBD) and meiotic maturation in the oocytes treated with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX).

A) The GVBD rate at 0, 0.5, 1, 2, 4, 8, and 12 h in IVM culture. Delayed GVBD was observed in the FSK and IBMX groups. B) The maturation rate at 22, 26, and 30 h after the initiation of IVM culture. Although delay in oocyte maturation occurred in the FSK and IBMX groups, the maturation rates at 30 h were comparable to those in the control groups. Error bars represent standard error of the mean. Bars labeled with different letters show significant differences (P < 0.05).

Fig 2. Transient upregulation of cAMP levels in vitrified-warmed germinal vesicle-stage (GV) oocytes treated with forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX).

A) Effect of FSK and IBMX on the level of intraoocyte cAMP during IVM culture. The intraoocyte cAMP level was reduced by vitrification prior to IVM culture, and this low cAMP level could be increased back to normal by supplementation of the IVM medium with FSK or IBMX. B) Level of intraoocyte cAMP at the initiation of culture (Group 1) or after culturing for 0.5 h (Group 2), immersion of vitrification/warming solution (Group 3), vitrification and warming (Group 4), vitrification and warming in vitrification/warming solution supplemented with FSK, IBMX or the combination of FSK and IBMX (Group 5–7). The decline in the cAMP level was induced by the vitrification procedure. Non-vitrification, GV oocytes cultured in mTCM199/20%SSS for 0.5 h; immersion, GV oocytes immersed in the vitrification/warming solution; vitrification, GV oocytes vitrified and warmed. Error bars represent standard error of the mean. Bars labeled with different letters show significant differences (P < 0.05).

Fig 3. Delayed MPF elevation induced by the treatment of vitrified-warmed GV oocytes with forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX).

Effect of FSK and IBMX on intraoocyte MPF activity during IVM culture. Low MPF activity was observed in the vitrified-warmed control group at the end of IVM culture, whereas the MPF activity of the vitrified-warmed FSK and IBMX groups was comparable to that of the corresponding fresh groups. Error bars represent standard error of the mean. Bars labeled with different letters show significant differences (P < 0.05).