Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF.
Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and apoptosis, a phenotype remarkably similar to that observed in oocytes and embryos from older women. A time-lapse video of a mouse oocyte imaged by polscope may be purchased for viewing on the internet at www.rbmonline.com/Article/824 (free to web subscribers).
Keefe
David
D
Department of Obstetrics and Gynecology, Division of Reproductive Medicine and Infertility, Women and Infants Hospital, 101 Dudley Street, Providence, RI 02905, USA. dkeefe@wihri.org
Liu
Lin
L
Wang
Wei
W
Silva
Celso
C
D000375:Aging; D000782:Aneuploidy; D000818:Animals; D006224:Cricetinae; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D006801:Humans; D008540:Meiosis; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D008941:Spindle Apparatus; D013816:Thermodynamics; D013997:Time Factors; D015044:Zona Pellucida / Q000473:pathology