Project description:Cumulative stress and adverse experiences precipitate changes in overall health, disrupting homeostasis and increasing disease. Stress is a known driver of allostatic changes, allowing for cellular adaptation in the face of environmental challenges. However, the molecular mechanisms regulating allostasis and long-term changes in intra- and intercellular signaling important for health are not clear. In males, chronic stress produces lasting changes in epididymal epithelial cell (EEC) intercellular signaling and extracellular vesicle (EV) composition important for sperm maturation. Here we used this system to assess the mechanisms regulating allostasis and the role of EVs to impact downstream target cell physiology and function. We found that prior corticosterone treatment decreased EEC energy requirements and altered mitochondrial ultrastructure, with changes in the cellular set point involving the mitochondrial complex I. CUT&RUN sequencing and gene co-expression network analysis separately identified significant epigenetic and transcriptomic reprogramming important for mitochondrial function. We found this new EEC allostatic state regulated EV intercellular communication with sperm, where EVs isolated from stress EECs increased sperm mitochondrial respiration, ultimately increasing sperm motility. These data support a signaling pathway by which a new cellular allostatic setpoint can be communicated to other cells, affecting their function.
Project description:Cumulative stress and adverse experiences precipitate changes in overall health, disrupting homeostasis and increasing disease. Stress is a known driver of allostatic changes, allowing for cellular adaptation in the face of environmental challenges. However, the molecular mechanisms regulating allostasis and long-term changes in intra- and intercellular signaling important for health are not clear. In males, chronic stress produces lasting changes in epididymal epithelial cell (EEC) intercellular signaling and extracellular vesicle (EV) composition important for sperm maturation. Here we used this system to assess the mechanisms regulating allostasis and the role of EVs to impact downstream target cell physiology and function. We found that prior corticosterone treatment decreased EEC energy requirements and altered mitochondrial ultrastructure, with changes in the cellular set point involving the mitochondrial complex I. CUT&RUN sequencing and gene co-expression network analysis separately identified significant epigenetic and transcriptomic reprogramming important for mitochondrial function. We found this new EEC allostatic state regulated EV intercellular communication with sperm, where EVs isolated from stress EECs increased sperm mitochondrial respiration, ultimately increasing sperm motility. These data support a signaling pathway by which a new cellular allostatic setpoint can be communicated to other cells, affecting their function.
Project description:Membrane integrity is essential in maintaining sperm viability, signaling, and motility, which are essential for fertilization. Sperm are highly susceptible to oxidative stress, as they are rich in sensitive polyunsaturated fatty acids (PUFA), and are unable to synthesize and repair many essential membrane constituents. Because of this, sperm cellular membranes are important targets of this process. Membrane Lipid Replacement (MLR) with glycerophospholipid mixtures (GPL) has been shown to ameliorate oxidative stress in cells, restore their cellular membranes, and prevent loss of function. Therefore, we tested the effects of MLR on sperm by tracking and monitoring GPL incorporation into their membrane systems and studying their effects on sperm motility and viability under different experimental conditions. Incubation of sperm with mixtures of exogenous, unoxidized GPL results in their incorporation into sperm membranes, as shown by the use of fluorescent dyes attached to GPL. The percent overall (total) sperm motility was increased from 52±2.5% to 68±1.34% after adding GPL to the incubation media, and overall sperm motility was recovered from 7±2% after H2O2 treatment to 58±2.5%)(n = 8, p<0.01) by the incorporation of GPL into sperm membranes. When sperm were exposed to H2O2, the mitochondrial inner membrane potential (MIMP), monitored using the MIMP tracker dye JC-1 in flow cytometry, diminished, whereas the addition of GPL prevented the decrease in MIMP. Confocal microscopy with Rhodamine-123 and JC-1 confirmed the mitochondrial localization of the dyes. We conclude that incubation of human sperm with glycerolphospholipids into the membranes of sperm improves sperm viability, motility, and resistance to oxidizing agents like H2O2. This suggests that human sperm might be useful to test innovative new treatments like MLR, since such treatments could improve fertility when it is adversely affected by increased oxidative stress.
Project description:Changes in protein subdomains through alternative splicing often modify protein-protein interactions, altering biological processes. A relevant example is that of the stress-induced up-regulation of the acetylcholinesterase (AChE-R) splice variant, a common response in various tissues. In germ cells of male transgenic TgR mice, AChE-R excess associates with reduced sperm differentiation and sperm counts. To explore the mechanism(s) by which AChE-R up-regulation affects spermatogenesis, we identified AChE-R's protein partners through a yeast two-hybrid screen. In meiotic spermatocytes from TgR mice, we detected AChE-R interaction with the scaffold protein RACK1 and elevated apoptosis. This correlated with reduced scavenging by RACK1 of the pro-apoptotic TAp73, an outcome compatible with the increased apoptosis. In contrast, at later stages in sperm development, AChE-R's interaction with the glycolytic enzyme enolase-alpha elevates enolase activity. In transfected cells, enforced AChE-R excess increased glucose uptake and adenosine tri-phosphate (ATP) levels. Correspondingly, TgR sperm cells display elevated ATP levels, mitochondrial hyperactivity and increased motility. In human donors' sperm, we found direct association of sperm motility with AChE-R expression. Interchanging interactions with RACK1 and enolase-alpha may hence enable AChE-R to affect both sperm differentiation and function by participating in independent cellular pathways.
