卵泡分离及活力检测

卵泡分离及活力 卵泡分离及活力检测 Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice Human Reproduction ，21（11 ）：2794～2800, Shee-Uan Chen , Chung-Liang Chien , Ming-Yih Wu , Tzu-Hsin Chen , Shu-Mei Lai , and Yu-ShihYang ，Chung-Wu Lin Department of Obstetrics and Gynecology, Department of Anatomy and Cell Biology and Department of Pathology, National Taiwan Experiment 1: analysis of follicle morphology To assess the integrity of follicles after freezing and thawing, we examined the follicle morphology by histology. The ovaries removed from female C57BL/6J mice were randomly allocated to four groups:non-frozen controls, DCV, conventional vitrification and slow freezing. Six samples from six different ovaries in each group were examined. The non-frozen controls and specimens after thawing were fixed in Bouin’s solution. The specimen were embedded in paraffin blocks and serially sectioned into 7-m slices. They were stained with haematoxylin and eosin (H&E) and observed with light microscopy (400). Follicles were classified as follows: (i) primordial follicles with one layer of flattened granulosa cells surrounding the oocyte; (ii)primary follicles with one layer of cuboid granulosa cells and (iii) secondary follicles with two or three layers of granulosa cells (Gougeon,1986 :Gougeon A (1986) Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1,81～87). Antral follicles were not included in this study.To avoid counting follicles more than once, in every third section,only follicles with a visible nucleus were counted (Cleary et al.,2001). The definition of normality of follicles was based on the crite-ria proposed by Lucci et al. (2004). The follicles classified as morphologically normal were those containing an intact oocyte surrounded by well-organized granulosa cells (Figure 2A and B). In contrast, follicles were classified as degenerate if they had one or more of the following aspects: a pyknotic oocyte nucleus, shrunken ooplasm or disorganized granulosa cells (Figure 2C and D). Experiment 2: examination of follicle viability to evaluate the viability of follicles, we used Trypan Blue stain for follicles isolated from frozen¨Cthawed ovarian tissues and non-froze controls. Five samples from five different ovaries in each group were evaluated. The ovaries in each group were cut into small pieces. They were transferred to a 5-ml centrifuge tube containing 1 ml of HTF medium with 10% FBS plus 200 IU/ml type I collagenase (Sigma). at 37C for 1 h and The ovarian tissues were incubated in 5% CO2 mechanically disrupted using a Pasteur pipette every 15 min. The digestion was terminated by adding 4 ml of HTF medium with 50% FBS. After centrifuging at 400 g for 5 min, the pellet was resuspended in 100 l of HTF medium and mixed with 40 l of 4% Trypan Blue (Sigma). The suspension was spread thinly in a Petri dish and covered with oil for examination with an inverted microscope (400). Follicles were scored as being alive if the oocyte and the surrounding granulosa cells were clear (Figure 3A and B). Follicles were scored as being non-viable if the oocyte or the surrounding granulosa cells had blue colora-tion, demonstrating the inability to exclude the dye (Figure 3C and D). Experiment 5: allogeneic orthotopic transplantation of ovaries We further investigated the pregnancy potential of frozen¨Cthawed an non-frozen ovaries after transplantation. Female C57BL/6J mice were randomly allocated into the four groups. Non-frozen and frozen-thawed ovaries were orthotopically transplanted into allogeneic female recipient mice of C57BL/6J-Tyr c-2J-J albino strain (Behringer, 1994).Fifteen mice with 30 different ovaries in each group were transplanted.The recipient female mice were oophorectomized by removing ovaries from ovarian bursae before transplantation. Three weeks after grafting,recipients were paired with fertile albino male mice of C57BL/6J-Tyr c-2J-J strain. Mating was confirmed by the presence of a vaginal plug, and then the female was housed individually. If no vaginal plug was seen,females were left in continuous pairings with males for 28 days. The pregnant female mice were allowed to litter, and the size of each litter was recorded. The pups have black eyes and fur (Figure 5). Mare lectin