Oocyte meiotic-stage specific differences in spindle disassembly and reassembly in response to temperature changes

Abstract

OBJECTIVE: The objectives of this study were to compare the effect of temperature changes on meiotic disassembly in mouse MII and TI stage oocytes and to compare the reassembly of the spindle in MII and TI oocytes following vitrification and warming. DESIGN: Experimental. MATERIALS AND METHODS: Oocytes were collected from B6SJL-F2 female mice injected with 5 IU eCG followed 44 h later with 5 IU hCG. Oocytes were collected 12 h post-hCG and denuded. The first experiment TI and MII oocytes were placed into drops of HTFH at 37° C (control), room temperature (RT) and 4° C for 0, 5, 30 and 60 min before visualization of the spindle with the Oosight™ image analysis software. In the second experiment, TI and MII oocytes were vitrified and warmed. Oocyte spindles were measured at 0, 30 and 60 min post warming at 37° C. Results were analyzed using Chi-square analysis and considered significant at P<0.002. RESULTS: At 0 h all TI and MII oocytes had visible spindles. There were no differences in spindles in TI and MII oocytes after 5 min at RT. After 30 min at RT, there was a significant difference in presence of spindles between TI and MII oocytes (100, 43%), respectively. After 1h at RT a significantly higher percentage of TI oocytes had a spindle compared to MII oocytes (100, 25%), respectively. After 5 min at 4° C, significantly more TI oocytes had spindles compared to MII oocytes (100, 22%), respectively. After 30 and 60 min at 4° C, there were no spindles in MII oocytes while 100% of TI oocytes had a visible spindle (P<0.0005). No spindles were present in MII oocytes at 0h post warming compared to 100% in TI oocytes. After 30 min of warming there was a significantly higher percentage of TI oocytes with a spindle compared to MII oocytes (100, 57%), respectively. After 60 min there were no differences between spindle assembly in TI and MII oocytes. CONCLUSIONS: TI oocytes have a polar body present, making it impossible to distinguish from a MII oocyte under a light microscope. Selection of MII oocytes is essential in the success of ICSI and oocyte cryopreservation. This study demonstrates a striking difference in microtubule depolymerization in MII and TI oocytes. Further experiments will look into the differences in spindle properties between different stages of meiosis as well as the effect of vitrifying TI stage oocytes.