Females of internally fertilizing species can significantly extend sperm lifespan and functionality during sperm storage. The mechanisms for such delayed cellular senescence remain unknown. Here, we apply current hypotheses of cellular senescence developed for diploid cells to sperm cells, and empirically test opposing predictions on the relationship between sperm metabolic rate and oxygen radical production in an insect model, the cricket Gryllus bimaculatus . Using time-resolved microfluorimetry, we found a negative correlation between metabolic rate (proportion of protein-bound NAD[P]H) and in situ intracellular oxygen radicals production in freshly ejaculated sperm. In contrast, sperm stored by females for periods of 1 h to 26 days showed a positive correlation between metabolic rate and oxygen radicals production. At the same time, stored sperm showed a 37 per cent reduced metabolic rate, and 42 per cent reduced reactive oxygen species (ROS) production, compared with freshly ejaculated sperm. Rank differences between males in ROS production and metabolic rate observed in ejaculated sperm did not predict rank differences in stored sperm. Our method of simultaneously measuring ROS production and metabolic rate of the same sample has the advantage of providing data that are independent of sperm density and any extracellular antioxidants that are proteins. Our method also excludes effects owing to accumulated hydrogen peroxide. Our results unify aspects of competing theories of cellular ageing and suggest that reducing metabolic rate may be an important means of extending stored sperm lifespan and functionality in crickets. Our data also provide a possible explanation for why traits of ejaculates sampled from the male may be rather poor predictors of paternity in sexual selection studies and likelihood of pregnancy in reproductive medicine.

Predicting infertility is central to reproductive biology, medicine and evolutionary biology. In-vitro studies suggest that oxidative sperm damage causes infertility. Oxidative sperm damage can be reduced via two fundamental pathways: the removal of oxygen radicals by antioxidants, or the interference with cell metabolism to reduce the formation of oxygen radicals. Oxidative damage protection of spermatozoa should evolve frequently, especially during female sperm storage. However, in-vivo evidence linking oxidative protection and fertility is rare. We show that the intra-sperm production rate of oxygen radicals and the sperm metabolic rate were reduced in female bedbugs, Cimex lectularius, compared to males, and females laid fertile eggs. Females became infertile when sperm oxygen radicals and sperm metabolic rate increased to male levels. Our results link female fitness to sublethal sperm damage, imply adaptive benefits of interfering with sperm metabolism and offer the hypothesis that polyandry may serve to replace low-quality sperm.

Significance: Redox reactions play important roles in both physiological and pathological processes, highlighting the importance of quantifying and localizing intracellular redox-active components. Most research has focused on direct investigation of reactive oxygen species (ROS). Intensity-based fluorescent methods are very sensitive and easy to use, but they lack specificity and can produce artifacts. In this article, we focus on synthetic sensors, describing experimental pitfalls associated with their use. We also present alternative methods for the detection of free radicals. Recent Advances: New approaches have been developed to overcome the main artifact of intensity-based methods: spurious changes in fluorescence intensity caused by oxidation. These new approaches are based on analytical measurements of the oxidized sensors or techniques that are not susceptible to oxidation, such as electron spin resonance and fluorescence lifetime-based methods. Regardless of the approach, the need for detection of ROS on the subcellular level, especially in the mitochondria, has motivated the development of new probes. Critical Issues: Flow cytometry systems and confocal microscopes are now available to the majority of biologists, and commercially available probes are, therefore, more widely used. The fact that these new applications are cited in thousands of publications makes these sensors even more attractive. Future Directions: The field of ROS detection by synthetic sensors continues to expand, bringing needed additional research to the development of robust techniques that are applicable both in vitro and in vivo . Antioxid. Redox Signal. 25, 520–533.