There have been a few questions on RG relating to the induction of endogenous ROS. Truth is, it's an easy thing to do in principle, e.g. add a high concentration of glucose, angiotensin II (if sperm express the receptor; I don't know), arsenite etc. However it really does depend on what you're trying to replicate. Is it in relation to disease, or a physiological redox signal? You're right not to use H2O2 to mimic endogenous ROS generation (it annoys me when people use H2O2 for this purpose). Some people would add NADPH, thinking they can increase Nox activity, which it does, but a lot of enzymes use NADPH as an electron donor, and as such you may potentially activate a lot of enzymatic systems with potentially unfavourable results unrelated to ROS.

If you can clarify further the actual purpose of your experiment it may assist in you getting the right help with your query. Remember, there is a massive difference between oxidant signalling and oxidative stress.

Hello, X/XO and menadione are exogenous sources of superoxide. It is not induction strictly speaking. A well known form of stress for sperm is heat stress. 

Menadione as a quinone works by directly giving out superoxide, not inhibiting the ETC. 

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You may also try altering the salinity, osmotic pressure and glucose level.

How about using buthionine sulphoximine (BSO) to prevent the GSH synthesis? xCT (cystine/ glutamate antiporter) promotes the uptake of cysteine, the rate limiting substrate for the synthesis of reduced form of glutathione. 

Article Modulation of oxidative stress as an anticancer strategy

Hi Zarka,

Try pyocyanin and measure ROS as we did (http://www.nature.com/cddis/journal/v7/n3/full/cddis2015409a.html OR https://www.researchgate.net/publication/296698867_Dengue-induced_autophagy_virus_replication_and_protection_from_cell_death_require_ER_stress_PERK_pathway_activation).

If it is toxic try titrating it. Best of luck!

-ED

Article Dengue-induced autophagy, virus replication and protection f...

First, think about these cells as completely unique. The motor, which drives the cells are evidently very similar or the same as bacterial flagellum. It need ATP and Ca.  And keep in mind, that in ejaculate prostate adds huge amount of citrate (10 mm or so)/ Citrate chelates Ca but serves, evidently as a substrate. Until the cells are in the proper place, they are immobilized and thus mitochondria are overcharged. That is when sperm cells can produce superoxide radicals, if they do.

You can use extended periods of centrifugation [see article: Reactive oxygen species measurement in human spermatozoa by flow cytometry using the fluorescent probe, 2', 7'-dichlorofluores-cein-diacetate (DCFH-DA), Lampiao et al]

or

you can also place the sample in a fridge for a while and this insult might cause endogenous ROS production. (PMID: 23865569 )