Assessment of a ne w cryoloop vitrif ication protocol in the cryopreservation of mouse mature oocytes

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Valentine Dixon
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1 , 3,, (,,, ) ED15 (15 %ethylene glycol, EG + 15 % dimethylsulphoxide, DMSO),,, ( EG) (DMSO) h, ;, %, h (87. 0 % % % vs 95 %, P > ;91. 3 % % % vs 90 %, P > 0. 05) ; (81. 3 % vs %, P > ;76. 1 % vs %, P > 0. 05) ED15, ; ; ; ; R A (2008) Assessment of a ne w cryoloop vitrif ication protocol in the cryopreservation of mouse mature oocytes W A N G Zeng2y an, H E Fang2f an g, S U N Zheng2y i, C H EN Yi n g De p artment of Obstet rics & Gy necolog y, Peki ng Union Medical College Hos pit al, Peki ng Union Medical College, Chi nese A ca dem y of Medical S ciences, B ei j i ng Abstract Objective : To assess the outcome of a new cryoloop vitrification protocol in t he cryopreservation of mouse MII oocytes. Methods : Frozen2t hawed oocytes were investigated against t he fresh mouse MII oocyte as control. Wit h t he cryoloop carrier, mo use M II oocytes were vit rified in vit rificatio n solutio n co nsisting of 15 % et hylene glycol ( EG), 15 % dimet hylsulp hoxide (DMSO), 5. 8 mg/ ml Ficoll 400 and mol/ L sucrose ( ED 15 p rotocol ). Af ter t hawing, t he survival rates were reco rded. So me t hawed oocytes and f resh oocytes were fixed, and t he microt ubules of t he spindles and chro mo so mes were t hen stained by immunofluorescent method. Confocal microscopy was used to reveal t he configurations of t he spindles and chromo somes. Most of the ot her survived oocytes were inseminated in vitro, t hen t he fertilization rates and blastocyst formation rates were compared wit h t hose of t he control group. Results : The oocytes vitrified by this protocol survived very well. The number of thawed oocytes wit h normal spindles and chro mo so mes was similar to t hat of t he f resh o nes. The fertilizatio n rates and blastocyst formation rates of t he thawed oocytes were also high, similar to those of the control group. Conclusion : The ED15 vitrification protocol has little deleterious affect on t he MII mouse oocytes survival rate, spindles and chromosomes. Key words : Oocytes ; Vitrification ; Cryoloop ; Spindle ; Mouse ( J Rep rod Med 2008 :17 (1) :38242) ; (1982),,,,. ( 3 )

2 , (), [1 ],,,,,,, H EPES 5 mg/ ml ( HSA, Quinn s, ) ( H TF, Quinn s, ) ; H TF ;,, 10 % ( SPS, Quinn s, ),5 %CO2 45 ( KM),( PMSG, ) (hcg, ) 10 IU, hcg h,, 25 G, 80 IU/ ml,37 1 min,,, 5 %CO2 : (1) : ; (2) :,, ; [2 ] ( FBS, Gibco, ), HSA FBS 1. : H EPES 5 mg/ ml HSA 10 % FBS H TF,7. 5 % ( v/ v) (DMSO) % (v/ v) ( EG), 15 % (v/ v) EG + 15 % (v/ v) DMSO mg/ ml Ficoll 400 (Sigma, ) mol/ L (Sigma, ) 2 min,, ; 3045 s 2. : 110 d, 510 s,0. 28 mol/ L 1,, 2 min ; mol/ L 2,, 3 min ; 3, 5 min, 10 %SPS H TF, 5 % CO2,1 h,,,,,,,,,,, h 3 : 2030, : 3. 6 % 30 min,0. 5 % Triton X21002PBS h,115 mol/ L + 1 % Triton X21002PBS 30 min,pbs 15 min,(fluoresce2 in isot hiocyanate, FITC, Sigma, ) 90 min,pbs 2,4,62222 (4 diamidino222p henylindole, DA PI, Sigma, ) 10 min, PBS 2, (, ),4 FITC, FITC, 488 nm,

40 2008 2 17 1 515535 nm DA PI, DA PI, 543 nm, 590630 nm, 1012 KM,,, 12 h,12 10 6 / ml 2,,8,5 SPSS12. 0, 2 Fisher 612, 601, 98. 2 %,,, (1), ( P > 0. 05) (1) 1 ( n) 20 18 (95. 0) 19 (90.

3 nm DA PI, DA PI, 543 nm, nm, 1012 KM,,, 12 h, / ml 2,,8,5 SPSS12. 0, 2 Fisher 612, 601, %,,, (1), ( P > 0. 05) (1) 1 ( n) (95. 0) 19 (90. 0) 0 h (87. 0) a 20 (91. 3) a 1 h (90. 9) a 20 (95. 4) a 2 h (90. 3) a 28 (93. 5) a :a : P > , , %,5 267, % ( P > 0. 05) (2) HSA , % ; FBS , %, (85. 2 %) ( P > 0. 05),( P > 0. 05) a :,12 ; b :,, ED15

4 (85. 2) 156 (75. 4) (81. 3) 267 (76. 1) :, P > 0. 05, [9 ] 1986, 200, [ 1 ],,,,,,, BSA (, ) [2 ],, ( ICSI), [10 ] HSA FBS,,,,,, EG DMSO,15 %( ED15), [3 ] 20 %,Ficoll 400 ;,,, [4 ] [5 ] %83. 9 %,85. 2 %,,,, ED15 + Ficoll + MII,, [ 4 ], Rienzi [6 ] Gook [7 ], ED15 + Ficoll +,,,,( 50 s) () % [ 8 ], ED mg/ ml Ficoll mol/ L ED mg/ ml Ficoll mol/ L, %,, [ 1 ] Jain J K,Paulson RJ. Oocyte cryopreservation[j ]. Fertil Ster2 il, 2006, 86[ Suppl 3 ] : [ 2 ] Carroll J, Wood MJ, Whittingham D G. Normal fertilization and development of frozen2thawed mouse oocytes : protective action of certain macromolecules[j ]. Biol Reprod, 1993, 48 (3) : [ 3 ] Cai XY,Chen GA,Lian Y,et al. Cryoloop vitrification of rab2 bit oocytes[j ]. Hum Reprod, 2005, 20 (7) : [ 4 ] Stachecki JJ, Munne S, Cohen J. Spindle organization after cryopreservation of mouse, human, and bovine oocytes[j ].

5 Reprod Biomed Online, 2004, 8 (6) : [ 5 ] Almeida PA,Bolton VN. The effect of temperature fluctua2 tions on the cytoskeletal organisation and chromosomal con2 stitution of t he human oocyte[j ]. Zygote, 1995, 3 (4) : [ 6 ] Rienzi L,Martinez F,Ubaldi F,et al. Polscope analysis of mei2 otic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures [ J ]. Hum Re2 prod, 2004, 19 (3) : [ 7 ] Gook DA,Osborn SM,Bourne H,et al. Fertilization of hu2 man oocytes following cryopreservation : normal karyotypes and absence of stray chromosomes[j ]. Hum Reprod, 994,9 (4) : [ 8 ] Lane M, Gardner D K. Vitrificaiton of mouse oocytes using a Nylon Loop [J ]. Mol Reprod Dev, 2001, 58 (3) : [ 9 ] Ghetler Y, Skutelsky E, Ben Nun I, et al. Human oocyte cryopreservation and the fate of cortical granules [ J ]. Fertil Steril, 2006, 86 (1) : [ 10 ] Larman M G,Sheehan CB, Gardner D K. Calcium2free vitrifica2 tion reduces cryoprotectant2induced zona pellucida hardening and increases fertilization rates in mouse oocytes[j ]. Repro2 duction,2006,131 (1) :53261.,,,,(),,, : (1),,,, (2) (3),,, (4),, (5),,() (6),,,,,,, : Science Nature Elsevier ; : (),, (),,,,,,,,( ),,( ),

Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model

: 31 1 2004 Kor J Fertil Steril, Vol 31, No 1, 2004, 3 1 2,, 1 2 3 3 3 3 3 3 3* Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model Kwan Cheal Yang 1, Hee-Gyoo Kang 2,Hoi-ChangLee 3,