Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy

Abstract

Objective: Meiotic spindles are crucial for normal chromosome alignment and separation of chromosomes during meiosis I and meiosis II. Traditional evaluation of the integrity spindles has been the immunofluorescent staining of tubulin. Recently, Polscope has emerged as a tool to view spindles in living oocytes. In this study we assess the spindle status of thawed oocytes after vitrification using Polscope and investigate their feasibility to replace immunofluorescent staining. Design: The Polscope was used to image spindles in living mouse oocytes before vitification. After thawing, the spindles of oocytes were imaged again and classified according to the spindles images. The results were compared with those by immunofluorescent staining of tubulin. Materials/Methods: The oocyte-cumulus complexes were collected from ICR mice superovulated by PMSG and hCG injections. The cumulus cells were removed by hyaluronidase. The MII oocytes were imaged by Polscope. Only oocytes with good spindle morphology were subsequently undergoing vitrification thawing protocol. The oocytes were vitrified with 5.5 M Ethylene Glycol- and 1 M sucrose-containing Dulbecco phosphate buffered saline. They were loaded into thin walled pulled straw and were stored at liquid nitrogen till thawing. After thawing, the oocytes were evaluated by Polscope again. The spindleimages were saved in computer for later analysis. The survived thawed oocytes were then fixed and incubated with anti-tubulin monoclonal antibody. Tubulin was stained by fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG and chromatin was stained with Hoechst 33258. Results: Spindle images by Polscope were calssified as three groups (Wang et al. ASRM, 2001). Group I oocytes had normal spindles. Group II oocytes had abnormal spindles. Group III oocytes had no image of spindles. Spindle morphology by immunofluorescent staining was classified as normal, reduced or disrupted. Totally, 100 MII oocytes were vitrified. After thawing, 79 oocytes were morphologically intact. Images by Polscope were classified as 26 in group I, 28 in group II and 25 in group III. With immunofluorescent staining, 25 were classified as having normal spindle morphology, 30 as having abnormal spindle morphology and 24 as having disrupted morphology. The chromosome pattern was compact in 50 oocytes, dispersed in 24 oocytes and absent in 5 oocytes. Conclusions: The spindles of frozen thawed oocytes can be assessed using Polscope and the image is well correlated to the results by traditional immunofluorescent staining. After vitrification and thawing, only 26 (33%) of 79 oocytes appear good and have good morphology of spindles. A portion (32%) of oocytes has relative good appearance but absent spindle images. Without Polscope or immunofluorescent stain to microtubulin, these cannot be selected out. It reflected the unavoidable cryo-injury to meiotic spindle and the imperative improvement of the protocol. Whether the spindles of the thawed oocytes can recover after some period of incubation will need further investigation. Supported by: This study was supported by grants from Chang Gung Memorial Hospital CMRP 891 and CMRP 949.