Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print July 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025627
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Uwe Paasch
Rakesh K. Sharma
Sonja Grunewald
Edward J. Mascha
H-J Glander
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Submitted November 18, 2003
Returned for revision December 11, 2003
Accepted July 21, 2004

Male Reproductive Tract


Cryopreservation and Thawing Is Associated with Varying Extent of Activation of Apoptotic Machinery in Subsets of Ejaculated Human Spermatozoa

Uwe Paasch , Rakesh K. Sharma , Akshay K. Gupta , Sonja Grunewald , Edward J. Mascha , H-J Glander , and Ashok Agarwal *

* To whom correspondence should be addressed. E-mail: agarwaa{at}ccf.org.

Abstract
We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8 and -9 activity, intact mitochondrial membrane potential ({Delta}{psi}m) and DNA- fragmentation in human spermatozoa. Eleven pools of cryopreserved and 8 pools of fresh semen samples were examined. Mature and immature fractions were separated on a 2-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and it's binding to Annexin V labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using carboxyfluorescein conjugated inhibitors, {Delta}{psi}m using a lipophilic cationic dye and DNA fragmentation by the terminal deoxynucleotidyl transferase - mediated dUTP nick end labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8 and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA-fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm both ANMB+ and ANMB- fraction showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm apoptosis was induced by activation of caspase-9 following loss of intact {Delta}{psi}m. These results demonstrate that selection of Annexin V negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during cryopreservation process.

Key words: Male Reproductive Tract • Apoptosis • Signal transducers • Sperm • Spermatogenesis


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