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A Method for Chronological Intravital Imaging of Bovine Oocytes during In Vitro Maturation

Published online by Cambridge University Press:  06 November 2008

Morten R. Petersen*
Affiliation:
Department of Large Animal Sciences, Section for Veterinary Reproduction and Obstetrics, Faculty of Life Sciences, Copenhagen University, Dyrlaegevej 68, 1870 Frederiksberg C, Copenhagen, Denmark
Michael Hansen
Affiliation:
Department of Plant Biology, Faculty of Life Sciences, Copenhagen University, Thorvaldsensvej 40, 1870 Frederiksberg C, Copenhagen, Denmark
Birthe Avery
Affiliation:
Department of Large Animal Sciences, Section for Veterinary Reproduction and Obstetrics, Faculty of Life Sciences, Copenhagen University, Dyrlaegevej 68, 1870 Frederiksberg C, Copenhagen, Denmark
Ingrid B. Bøgh
Affiliation:
Department of Large Animal Sciences, Section for Veterinary Reproduction and Obstetrics, Faculty of Life Sciences, Copenhagen University, Dyrlaegevej 68, 1870 Frederiksberg C, Copenhagen, Denmark
*
Corresponding author. E-mail: mmpe@life.ku.dk
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Abstract

Oocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.

Type
Multiphoton Microscopy–Special Section
Copyright
Copyright © Microscopy Society of America 2008

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References

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