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Hum. Reprod. Advance Access published online on May 2, 2006
Human Reproduction, doi:10.1093/humrep/del109
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received December 15, 2005
Revised March 8, 2006
Accepted March 16, 2006

Article

Caspase-3, TUNEL and ultrastructural studies of small follicles in adult human ovarian biopsies

Peter R. Hurst 1 *, Jocelyn M. Mora 1, and Mark A. Fenwick 1

1 Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin, New Zealand

* To whom correspondence should be addressed.
Peter R. Hurst, E-mail: peter.hurst{at}stonebow.otago.ac.nz


  Abstract

BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles.

Keywords: apoptosis/caspase-3/follicles/human/ovary.
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