Maturation, Fertilization, and the Structure and Function of the Endoplasmic Reticulum in Cryopreserved Mouse Oocytes1

Abstract

Oocyte cryopreservation is a promising technology that could benefit women undergoing assisted reproduction. Most studies examining the effects of cryopreservation on fertilization and developmental competence have been done using metaphase IIstage oocytes, while fewer studies have focused on freezing oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation. Herein, we examined the effects of vitrifying GVstage mouse oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes necessary for proper fertilization and early embryonic development. We examined the endoplasmic reticulum (ER) as one indicator of cytoplasmic structure, as well as the ability of oocytes to develop Ca 2+ release mechanisms following vitrification and in vitro maturation. Vitrified GV-stage oocytes matured in culture to metaphase II at a rate comparable to that of controls. These oocytes had the capacity to release Ca 2+ following injection of inositol 1,4,5-trisphosphate, demonstrating that Ca 2+ release mechanisms developed during meiotic maturation. The ER remained intact during the vitrification procedure as assessed using the lipophilic fluorescent dye DiI. However, the reorganization of the ER that occurs during in vivo maturation was impaired in oocytes that were vitrified before oocyte maturation. These results show that vitrification of GV-stage oocytes does not affect nuclear maturation or the continuity of the ER, but normal cytoplasmic maturation as assessed by the reorganization of the ER is disrupted. Deficiencies in factors that are responsible for proper ER reorganization during oocyte maturation could contribute to the low developmental potential previously reported in vitrified in vitro-matured oocytes.assisted reproductive technology, gamete biology, in vitro fertilization, meiosis

Factors Affecting Cryopreservation of Porcine Oocytes

“…Besides mitochondria, the smooth endoplasmic reticulum is considered as the major regulator of Ca 2+ in oocytes. In mice, cryopreservation has been found to reduce oocyte development via affecting the function of endoplasmic reticulum [57]; however, such alterations and their consequences in vitrified porcine oocytes have not yet been investigated. Also meiotic spindle has been found to be frequently damaged by vitrification in matured porcine oocytes [21, 24,40,54].…”

A Comparison of Cryotop and Solid Surface Vitrification Methods for the Cryopreservation of In Vitro Matured Bovine Oocytes

“…Taken together with the high survival rates, this suggests that the impaired developmental ability of vitrified oocytes was caused by sub-lethal damage that could be mainly related to the cooling and/or warming procedure. Such sub-lethal damage may include disruption of the meiotic spindle and other cytoskeletal elements [10,37], damage and dysfunction of organelles such as mitochondria or endoplasmic reticuli [38][39][40] or degradation of cytoplasmic mRNA levels [41], which have been reported to occur in cryopreserved mammalian oocytes. Further improvements of the present vitrification protocols will be necessary to avoid such damage.…”

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

“…Developmental arrest was observed more frequently in vitrified group than fresh control group ( Table 1), suggesting that oocyte activation by sperm penetration was suboptimal in the vitrified-warmed oocytes. Vitrification induced the reduction of maturation promoting factor (MPF) in ovine oocytes before fertilization [34], the damage of mitochondria in bovine oocytes [35], and of endoplasmic reticulum in mouse oocytes [36]. Post-warm oocytes may have been recovered by 2-h culture prior to IVF in the present study, but the harmful effect of remaining intracellular CPA (EG and/or DMSO) could not be estimated.…”