title: Effects of chilling on structural aspects of early preantral mouse follicles.
abstract: Chilling injury is one of the major limiting factors for achieving optimal cryopreservation of gametes. This study aimed to determine potential chilling-induced damage on several structural aspects of early preantral mouse follicles. Mechanically isolated intact early preantral follicles (type 3b-4) were exposed to 0 degrees C for 1, 5, 10, or 30 min. Control and chilled follicles were analyzed by confocal microscopy after staining for tubulin, F-actin, and chromatin, and by electron microscopy. Chilling for only 1 min was sufficient to cause depolymerization of microtubules in the oocyte and the surrounding granulosa cell layer as evidenced by a substantial decrease in fluorescence intensity after antitubulin labeling. Cooling for longer periods caused alterations in microtubule organization in the follicle-enclosed oocyte. These alterations included the loss of interphase microtubules, concomitant with the formation of perinuclear or cortical microtubule asters and sometimes a complete disappearance of microtubules. The extent of microtubule modification was related to the time of chilling, but was fully reversible after rewarming follicles at 37 degrees C for 1 h. Chilling had only minor effects on the actin-containing elements located predominantly in the oocyte cortex and the transzonal projections. Ultrastructural analysis confirmed that oocyte-somatic cell interactions were present. There was no influence on the chromatin configuration within the follicle-enclosed oocyte. These results indicate that mouse follicles are relatively tolerant to direct chilling injury and, as a consequence, are able to withstand the cooling-warming steps during conventional cryopreservation procedures.
authors.affiliation: Follicle Biology Laboratory, University Hospital and Medical School, Vrije Universiteit Brussel, B-1090 Brussels, Belgium. leen.vanhoutte@ugent.be
authors.forename: Leen
authors.initials: L
authors.lastname: Vanhoutte
authors.forename: Rita
authors.initials: R
authors.lastname: Cortvrindt
authors.forename: Daniela
authors.initials: D
authors.lastname: Nogueira
authors.forename: Johan
authors.initials: J
authors.lastname: Smitz
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D002843:Chromatin / Q000648:ultrastructure; D003080:Cold Temperature; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D015739:Nocodazole / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D006080:Ovarian Follicle / Q000033:anatomy & histology* / Q000187:drug effects / Q000648:ultrastructure; D014404:Tubulin / Q000378:metabolism / Q000502:physiology
chemical_list: D000199:Actins; D002843:Chromatin; D019384:Nucleic Acid Synthesis Inhibitors; D014404:Tubulin; D015638:Cytochalasin D; D015739:Nocodazole