Histological and ultrastructural analysis of cryopreserved sheep preantral follicles.
The aim of this study was to verify the histological and ultrastructural characteristics of sheep preantral follicles after exposure of ovarian tissue to cryopreservation in glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) in order to determine the optimum method to store sheep ovarian tissue for later experimental or clinical use. Each ovarian pair from five mixed-breed ewes was divided into 17 fragments. One (control) fragment was immediately fixed for routine histological and ultrastructural studies and the remaining (test) fragments were randomly distributed in cryotubes, equilibrated at 20 degrees C/20 min in 1.8 mL of minimal essential medium (MEM) containing 1.5 or 3 M GLY, EG, PROH or DMSO and then either fixed for morphological studies to determine their possible toxic effect or frozen/thawed and then fixed to test the effect of cryopreservation on preantral follicles. Histological analysis showed that, compared to control fragments, all cryoprotectants at both concentrations significantly reduced the percentage of normal preantral follicles in ovarian fragments prior to or after cryopreservation. PROH 3.0 M appeared to exert a more toxic effect (P<0.05) than the other cryoprotectants in noncryopreserved tissues. After freezing/thawing, the highest (P<0.05) percentages of lightmicroscopical normal preantral follicles were observed in ovarian fragments cryopreserved in EG (1.5 and 3 M) or DMSO (1.5 M). However, transmission electronic microscopical (TEM) examination showed that only the DMSO-cryopreserved preantral follicles had normal ultrastructure. The data suggest that sheep preantral follicles should be cryopreserved with 1.5 M DMSO for later clinical or experimental application.
Laboratory of Manipulation of Oocytes Enclosed in Preantral Follicles--LAMOFOPA, Faculty of Veterinary, State University of Ceará, Fortaleza, CE, Brazil. regianers_2002@yahoo.com.br
Regiane R
RR
Santos
Ana P R
AP
Rodrigues
Sonia H F
SH
Costa
José R V
JR
Silva
Maria H T
MH
Matos
Carolina M
CM
Lucci
Sonia N
SN
Báo
Rob
R
van den Hurk
José R
JR
Figueiredo
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents; D003593:Cytoplasm / Q000648:ultrastructure; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D005260:Female; D005990:Glycerol; D006358:Hot Temperature; D008854:Microscopy, Electron; D006080:Ovarian Follicle / Q000648:ultrastructure*; D010053:Ovary / Q000648:ultrastructure; D011409:Propylene Glycols; D012756:Sheep; D014021:Tissue Preservation / Q000662:veterinary*
D003451:Cryoprotective Agents; D011409:Propylene Glycols; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide