Effect of cumulus cells and vitrification protocol on survival and subsequent development. Golestan jahromi PhD student. Introduction . Several lines of evidence indicate that surrounding cumulus cells play a fundamental role in the maturation process and full development competence .
Effect of cumulus cells and vitrification protocol
on survival and subsequent development
Several lines of evidence indicate that surrounding cumulus cells play a fundamental role in the maturation process and full development competence
cumulus cells are beneficial to oocyte survival after cryopreservation
may minimize the release of cortical granules prevent premature zona hardening
maintaining fertilization capacity of cryopreserved oocytes
Chian et al. reported that bovine oocytes matured without cumulus cells had a higher survival rate after vitrification.
Moreover, the rate of embryo development to the 8-cell stage in cumulus-cells free oocytes was significantly higher than that of cumulus cell-intact oocytes.
Ice growth and recrystallization are considered to be important factors in determining vitrification outcomes.
Synthetic ice blockers, which specifically inhibit the formation/emergence of ice nuclei and ice crystal growth, have recently been used to supplement vitrification solutions
Unlike conventional cryoprotectants that inhibit freezing by interacting with water, ice blockers are believed to bind to the surface of growing ice crystals and inhibit the addition of any further water molecules in specific planes of growth
This selective attraction to surfaces of ice growth permits ice blockers to exert significant effects even while present at very low concentrations.
Small quantities of ice blocker can therefore modify the number and size of ice crystals and thereby change the vitrification tendency of a solution without adding additional toxicity
The commercially available ice blockers are SuperCool X-1000 and SuperCool Z-1000.
To evaluate the effect of the presence of cumulus cells on the outcome of vitrification of GV or MII oocytes
The effect of adding ice blockers SuperCool X-1000 and SuperCool Z-1000 to vitrification media on oocyte survival and subsequent embryonic development
According to the manufacturer, the cooling and warming rates of the Cryotop are 23,000 and 42,000 °C/min, respectively
Motile spermatozoa were obtained by centrifugation of frozen–thawed semen
Day 2 frozen–thawed semen
Day 8 after
proportion of oocytes
developing to the blastocyst stage
VS +1% (v/v)
VS +1% (v/v) X-1000
VS +1% (v/v)
Z-1000 and 1% (v/v) X-1000
The data for survival, cleavage and blastocyst rates were expressed as mean ± SD and analyzed using one-way ANOVA.
Differences were considered significant at a level of P < 0.05.
Results frozen–thawed semen
Cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher cleavage rate and blastocyst rate than those vitrified at MII stage (P < 0.05).
The role of the cumulus cells during vitrification of MII oocytes remains controversial.
Some investigators reported that cumulus presence would protect MII oocytes against vitrification-induced damage.
Zhang et al. found no difference in the development of vitrified ovine MII oocytes with or without cumulus cells.
Gasparrini et al. reported that the presence of cumulus cells severely reduced the cleavage rate of MII buffalo oocytes following vitrification
It is generally accepted that cumulus-oocyte communication via an intact corona radiata is necessary for oocytes to attain full cytoplasmic maturation during IVM and improve fertilization rates during IVF
we established that the cleavage rate of denuded (GV and MII) bovine oocytes was significantly reduced compared to cumulus-enclosed oocytes, and almost no denuded bovine oocytes developed up to blastocyst stage after in vitro fertilization.
The survival, cleavage and blastocyst rate of cumulus-enclosed vitrified oocytes are significantly higher than that of partially-denuded vitrified oocytes.
In the present study:
No significant differences were detected between vitrified cumulus-enclosed and partially-denuded oocytes in the survival, cleavage and blastocyst rate.
The possible explanation is the cumulus was detrimental to vitrification, which comprises the benefits of cumulus in IVF procedure.
From another point of view, the intracytoplasmic sperm injection technique rather than conventional IVF has been used to achieve fertilization, which can circumvent the detrimental effects of removing the cumulus on subsequent zona penetrability.
The cell cycle stage during meiosis appears to affect the results of bovine oocyte vitrification due to varying sensitivity to cooling procedures.
Chilling injury is reported to be higher in vitrified immature oocytes, owing to low membrane stability and susceptibility of the cytoskeleton
However, an increase in chromosomal abnormality has been observed in vitrifiedmature oocytes, owing to alterations in the meiotic spindle.
The results indicate that cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher cleavage and blastocyst rate than those vitrified at MII.
This may be due to the increase in volume associated with cumulus expansion during maturation.
It may also be due to the higher water permeability (Lp) and solute permeability (Ps) of MII than GV bovine oocytes.
That means the changes of cell volume and intracellular CPA concentrations are more severe in MII than GV bovine oocytes during CPA addition and dilution process, which make it more sensitive.
In the present study, we report for the first time the effect of ice blockers on the bovine oocytes, however, the results indicate that the survival rate and development competence of bovine oocytes vitrified in solutions supplemented with or without X-1000 and/or Z-1000 by Cryotop method are not significantly different.
Ice blockers did not affect the survival rate and developmental competence of vitrified bovine oocytes.
When the vitrification systems are large volume, such as organs, in which a large quantity of nucleators exit, ice blockers can suppress nucleation and recrystallization by binding to nucleators in solutions during vitrification and warming. Therefore, the ice growth was inhibited and damage to the systems was reduced.
In this study, the combination of the two ice blocker agents inhibit blastocyst development maybe because 1% X-1000 and 1% Z-1000 is not the ideal balance, which shows less effective than either agent alone.
cumulus-enclosed GV bovine oocytes survived exhibited a significantly higher cleavage rate and blastocyst rate than those vitrified at MII stage (P < 0.05). vitrification and subsequently developed at higher rates than MII oocytes .
Ice blockers had no effect on bovine oocytevitrification.