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2. Effect of cumulus cells and vitrification protocol on survival and subsequent development Effect of cumulus cells and vitrification protocol on survival and subsequent development Golestan jahromi PhD student Golestan jahromi PhD student

3. Introduction Several lines of evidence indicate that surrounding cumulus cells play a fundamental role in the maturation process and full development competence

4. Introduction 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

5. Introduction 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.

6. Introduction The need to maintain cumulus cells during cryopreservation of oocytes is still a matter of debate. Considering that the functions of cumulus cells are different in immature (GV) and mature (MII) oocytes, it is necessary to determine the effects of cumulus cells on bovine oocyte vitrification at both the GV and the MII stage, respectively

7. Introduction Water permeability (Lp), solute permeability (Ps), and activation energies for Lp and Ps (Ea) were determined using a two parameter model. Osmotic tolerance limits and toxic tolerance limits were determined based on oocyte developmental competence following exposure to different CPAs.

8. Introduction 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

9. Introduction 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.

10. Introduction 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

11. Introduction The commercially available ice blockers are SuperCool X-1000 and SuperCool Z-1000.

12. The SuperCool X-1000 is a copolymer of polyvinyl alcohol (PVA) of mean molecular mass 2000 Da, with 20% of the hydroxyls replaced by acetate groups. The SuperCool Z- 1000 is a copolymer of polyglycerol (PGL) of mean molecular mass 750 Da

13. 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

15. Collection of oocytes IVM Vitrification warming IVF

16. In vitro maturation Groups of 50 COCs were placed in 500 μl maturation medium in four-well plates and cultured for 24 h at 39 °C in a 5% CO2 humidified air atmosphere. The maturation medium was Tissue Culture Medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS) and 10 ng/ml epidermal growth factor (EGF).

17. Vitrification and warming The holding medium (HM) used for handling oocytes during vitrification and warming was HEPES-buffered TCM 199 supplemented with 20% (v/v) FBS. All manipulations were performed on a 39 °C heated stage in a warm room (25–27 °C). All the media were used at room temperature, except for the warming solution which was used at 37 °C.

18. According to the manufacturer, the cooling and warming rates of the Cryotop are 23,000 and 42,000 °C/min, respectively

19. Control and vitrified GV and MII oocytes were fertilized using the same conditions. All oocytes were washed four times in fertilization medium, and then transferred in groups of 50 into four-well dishes containing 250 μl of fertilization medium (Tyrode’s medium with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate and 6 mg/ml fatty acid-free BSA). In addition, 10 μg/ml heparin–sodium salt was added

20. Motile spermatozoa were obtained by centrifugation of frozen–thawed semen

21. Zygotes were transferred in groups of 25 to 25 μl culture droplets of synthetic oviductal fluid medium (SOF) under mineral oil

22. Day 2 after insemination Day 8 after insemination Cleavage ratesproportion of oocytes developing to the blastocyst stage

23. Experiment 1 COCsGV Cumulus- enclosed partially- denuded oocytes MII Cumulus- enclosed partially- denuded oocytes

24. Experiment 2: Effect of ice blocker X-1000 and Z-1000 COCsControlBasic media Basic media + X-1000 Basic media + Z-1000 Basic media + X-1000 + Z- 1000 VS +1% (v/v) X- 1000 VS +1% (v/v) Z-1000 VS +1% (v/v) Z-1000 VS +1% (v/v) Z-1000 and 1% (v/v) X-1000 VS +1% (v/v) Z-1000 and 1% (v/v) X-1000

25. Statistical analysis 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.

26. Results

27. development of bovine GV stage Oocytes treated NSurvived, n (%) Cleaved, n (%) Blastocyst, n (%) Blastocyst/cl eavage (%) Cumulus- enclosed control 141 (100 ± 0.0) 121 (86.3 ± 1.9) 47 (33.8 ± 1.8) (39.5 ± 2.2) Partially- denuded control 118117 (99.3 ±0.8) 89 (75.8 ± 3.9) 14 (11.5 ± 4.2) (14.8 ± 5.2) Cumulus- enclosed vitrified 177166 (93.8 ± 2.5) 108 (65.8 ± 5.6) 19 (11.3 ± 1.7) (18.0 ± 3.5)b Partially- denuded vitrified 143117 (81.3 ± 3.6)c 56 (47.3 ± 4.0)c 4 (4.0 ± 2.3)c (7.8 ± 4.5)b

28. embryo development of bovine MII stage Oocytes treated NSurvived, n (%) Cleaved, n (%) Blastocyst, n (%)Blastocyst/cl eavage (%) Cumulus- enclosed control 130126 (96.8 ± 1.5)a 115 (91.6 ± 2.5)a 45 (35.6 ± 2.8)a(39.0 ± 4.1)a Partially- denuded control 122119 (97.4 ± 1.1)a 92 (78.4 ± 4.7)b 32 (27.2 ± 2.3)b(34.6 ± 2.7) a,b Cumulus- enclosed vitrified 158147 (93.0 ± 2.3)a 51 (35.2 ± 4.6)c 7 (5.0 ± 4.3)c(12.6 ± 9.7) b,c Partially- denuded vitrified 167153 (91.8 ± 2.4)a 57 (36.8 ± 3.2)c 7 (4.4 ± 1.4)c(10.8 ± 3.5)c

29. development of bovine GV stage cumulus-enclosed oocytes vitrified with different ice blocker media Oocytes treated N Survived, n (%) Cleaved, n (%)Blastocyst, n (%) Blastocyst/cle avage (%) Control 110104 (94.4 ± 1.9)a 77 (74.6 ± 3.2) 24 (23.0 ± 10.4) (31.2 ± 1.9) Basic media 11498 (86.0 ± 2.7)b 38 (38.0 ± 3.2) 2 (2.0 ± 1.3)(6.2 ± 4.1) Basic media + X-1000 11197 (88.4 ±2.9) 36 (37.2 ± 2.4) 3 (2.8 ± 1.2)(7.8 ± 3.2) Basic media + Z-1000 11093 (83.6 ± 4.8) 37 (40.0 ± 3.0) 2(2.2 ± 1.4)(4.8 ± 3.0) Basic media + X-1000 + Z-1000 114101 (88.0 ± 3.6) 43 (41.4 ± 6.7) 0 (0)(0)

30. 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).

31. Why?

32. Discussion 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.

33. Discussion 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

34. Discussion 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

35. Discussion 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.

36. In other species, the effect of cumulus cells on immature oocytes following vitrification was evaluated by the maturation rate, spindle and chromatin quality.

37. Discussion Tharasanit et al. [33] reported that cumulus removal from equine oocytes prior to IVM or vitrification resulted in reduced meiotic competence, MII spindle and chromatin quality. while the cumulus does protect immature equine oocytes during vitrification it does so by mechanisms other than support during maturation

38. Discussion In contrast, Bogliolo et al. reported that immature ovine oocytes vitrified without cumulus cells showed a significantly higher survival and meiotic maturation rate than those with cumulus cells, and no differences in spindle and chromatin organization between two groups were observed.

39. In this study: The survival, cleavage and blastocyst rate of cumulus-enclosed vitrified oocytes are significantly higher than that of partially-denuded vitrified oocytes.

40. Kuwayama et al. reported higher survival and blastocyst rate for cumulus-enclosed than cumulus-free human MII oocytes after vitrification

41. Tharasanit et al. found that cumulus-enclosed equine MII oocytes preserved their meiotic spindle and chromatin quality better during vitrification than denuded oocytes. Conversely, other investigators believed cumulus cells are not necessary or detrimental to MII oocytes during vitrification.

42. Discussion In the present study: No significant differences were detected between vitrified cumulus-enclosed and partially-denuded oocytes in the survival, cleavage and blastocyst rate.

43. Discussion 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.

44. Discussion Moreover, the oocytes were easier to handle for vitrification using a Cryotop when the oocytes were denuded completely from their cumulus cells. Therefore, cumulus removal prior to vitrification is at present a standard practice during cryopreservation of MII human oocytes.

45. Discussion 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

46. Discussion However, an increase in chromosomal abnormality has been observed in vitrified mature oocytes, owing to alterations in the meiotic spindle.

47. Discussion 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.

48. Discussion 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.

49. Discussion The osmotic tolerance limits tests indicated that the safe volume changes of GV bovine oocytes were from 150% to 46% of their volume. Toxicity tolerance limits tests suggested that Me2SO are more toxic than EG, and the exposure time to 30% and 40% EG should be less than 1 min.

50. Discussion 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.

51. Discussion Ice blockers did not affect the survival rate and developmental competence of vitrified bovine oocytes.

52. Why?

53. Discussion 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.

54. Discussion 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.

55. 55 Conclusion cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes. Ice blockers had no effect on bovine oocyte vitrification.