Reproduction Abstracts (2014) 1 P012 | DOI: 10.1530/repabs.1.P012

Mitochondrial activity and reactive oxygen species production define distinct subpopulations of human sperm with different functional properties

João Ramalho-Santos1, Mónica Marques1, Artur Paiva2, Teresa Almeida-Santos3 & Ana Paula Sousa3

1Center for Neuroscience and Cell Biology, Coimbra, Portugal; 2Portuguese Institute for Blood and Transplantation, Coimbra, Portugal; 3Human Reproduction Service, University Hospitals of Coimbra, Coimbra, Portugal.

Human ejaculates are heterogeneous, and can be comprised of different subpopulations. We have shown that mitochondrial activity varied within ejaculates, and that sperm with high mitochondrial activity were more functionally relevant. Mitochondria are also known to produce reactive oxygen species (ROS), which have been implicated both in sperm function, and in causing gamete dysfunction, depending on amounts and timing of production. We have applied several fluorescent probes in order to quantify ROS production in human sperm samples using flow cytometry. We show that human ejaculates are heterogeneous in terms of ROS production, with several subpopulations clearly detectable, comprised of sperm that produce increasing amounts of ROS (ROS−, ROS+, ROS++, etc.). In the case of the probe MitoSOX™ Red (MitoSOX), the sperm subpopulation producing the lowest amount of ROS represented the most functional subset of male gametes within the ejaculate, as it was correlated with the highest amount of live and non-apoptotic sperm, and increased both in samples with better semen parameters, and in samples processed by both density gradient centrifugation and swim-up, both known to select for higher quality sperm. Importantly the MitoSOX ROS− subpopulation was clearly more prevalent in samples that gave rise to pregnancies following Assisted Reproduction, regardless of other characteristics. Currently we are characterizing human sperm subpopulations with distinct mitochondrial properties at the molecular level. Our work therefore suggests that mitochondrial function may represent a strategy to both evaluate sperm samples, and isolate the most functional gametes for Assisted Reproduction.

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