Noninvasive imaging of spindle dynamics during mammalian oocyte activation.
OBJECTIVE
To develop a method to evaluate spindle dynamics in living oocytes and in karyoplasts during the initial stages of activation and after pharmacological disruption of cytoskeleton.


METHODS
Morphological study using a novel microscope.


METHODS
Translational research laboratory at marine biological laboratory.


METHODS
Six-week-old CD-1 or B6C3F1 mice superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG).


METHODS
Spindles of living oocytes and karyoplasts were imaged at 5-10 minute intervals using the Pol-Scope during the initial stages of oocyte activation and after pharmacological disruption of cytoskeleton.


METHODS
Assessment of spindle dynamics using Pol-Scope imaging.


RESULTS
During oocyte activation, spindle mid-region birefringence increased, followed by spindle rotation and second polar body extrusion in both intact oocytes and karyoplasts. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate failed to induce spindle activation in 60% of living oocytes and caused spindle disruption in some oocytes. Inhibition of PKC by a myristoylated PKC pseudosubstrate inhibited metaphase II release in most oocytes evaluated (86.7%). Cytochalasin D inhibited only spindle rotation and separation. Nocodazole disrupted spindles in less than 5 minutes after administration.


CONCLUSIONS
Pol-Scope imaging allows investigation at near real time of spindle dynamics during activation of living oocytes. Spindles also showed evidence of activation even in karyoplasts. The procedure may be useful for detecting functional spindle aberrations in living oocytes. Further studies are needed to determine whether spindle dynamics predict clinical outcome.
Navarro
Paula A A S
PA
Department of Obstetrics & Gynecology, Women & Infants Hospital, Brown University, Providence, Rhode Island, USA.
Liu
Lin
L
Trimarchi
James R
JR
Ferriani
Rui A
RA
Keefe
David L
DL
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002843:Chromatin / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology; D005260:Female; D008540:Meiosis / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods*; D015739:Nocodazole / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D008941:Spindle Apparatus / Q000502:physiology* / Q000648:ultrastructure; D013194:Staining and Labeling; D013324:Strontium / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014404:Tubulin / Q000502:physiology
D000970:Antineoplastic Agents; D002273:Carcinogens; D002843:Chromatin; D019384:Nucleic Acid Synthesis Inhibitors; D014404:Tubulin; D015638:Cytochalasin D; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D015739:Nocodazole; D013324:Strontium