卵泡分离及活力检测.docVIP

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 (HE) 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