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BOR - Papers in Press, published online ahead of print December 10, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020933
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BIOLOGY OF REPRODUCTION 70, 1041–1048 (2004)
DOI: 10.1095/biolreprod.103.020933
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Effects of Chilling on Structural Aspects of Early Preantral Mouse Follicles1

Leen Vanhoutte2,3, Rita Cortvrindt, Daniela Nogueira, and Johan Smitz

Follicle Biology Laboratory, University Hospital and Medical School, Vrije Universiteit Brussel, B-1090 Brussels, Belgium

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°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°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.

1 Supported by grants from the Ministry of Economical Affairs of Brussels Capital (grant RIB-2001/12).

2 Correspondence: Leen Vanhoutte, Follicle Biology Laboratory, University Hospital and Medical School Infertility Center, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. FAX: 0032 2 477 50 60; leenvanhoutte{at}hotmail.com; leen.vanhoutte{at}ugent.be

3 Current address: Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium






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Copyright © 2004 by the Society for the Study of Reproduction.