The immobilization of human spermatozoa bySTAT3 inhibitory compound V results from an excessive intracellular amount of reactive oxygen species

Abstract

We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca 2+ concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species (ROS). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress. To address this question, spermatozoa were incubated with Stattic V in combination with a caspase inhibitor, a proteasome inhibitor or a cell permeant ROS scavenger. Following incubation in different conditions, sperm motility was evaluated by CASA, acrosomal integrity by FITC conjugated Pisum sativum agglutinin (PSA-FITC) labeling, intracellular pH, and mitochondrial superoxide production by flow cytometry using BCECF and MitoSoxRed dye, respectively. Levels of reduced thiols were assessed by iodoacetamidofluorescein staining on total and on sperm surface proteins, and protein tyrosine phosphorylation was evaluated by western blot. The loss in sperm motility induced by Stattic V was associated with a slight intracellular acidification and an important increase in intracellular superoxide anion. Unlike caspase and proteasome inhibitors, low molecular weight thiols, such as N-acetyl-L-cysteine (NAC), prevented Stattic V-induced sperm immobilization and increase responsiveness to acrosome reaction inducers. NAC also efficiently prevented the production of superoxide anion, mitochondrial membrane depolarization, intracellular acidification and the oxidation of protein free thiols caused by Stattic V. These results show that the deleterious effects of Stattic V on sperm functions are caused directly or indirectly by excessive intracellular ROS production without causing sperm apoptosis or necrosis.

Metabolic enhancers supporting 1-carbon cycle affect sperm functionality: an in vitro comparative study

“…Actually, the fact that the NAC exhibits an isoelectric point equal to 5, makes the treatment more effective in acidic environments, canceling its effects under the typical alkaline conditions of ascidian sperm. In the human, previous studies 57 found that 0.5 mM NAC prevented sperm immobilization induced by Stat3 inhibitory compound V (Stattic V) as well as the production of superoxide anion, mitochondrial membrane depolarization and the oxidation of protein free thiols caused by Stattic V; however, with regards to pH i , 0.5 mM NAC did not significantly affect pH i but efficiently counteracted Stattic V-induced acidification.…”

Oxidative Stress and Male Fertility: Role of Antioxidants and Inositols

“…In vitro studies evaluated the effect of NAC on human spermatozoa using Stattic V, a non-peptidic inhibitor of STAT3 involved into sperm function [84,85]. Stattic V is associated to sperm apoptosis and sperm immobilization due to increase of different pathological outcomes such as acrosome reaction, intracellular Ca 2+ concentration, extracellular levels of reactive oxygen species (ROS), mitochondrial membrane depolarization and decrease in sperm ATP content [86]. Other in vivo study assessed the effect of 600 mg/day of NAC for three months to evaluate different parameters as chromatin negative alteration induced by high oxidative stress and its consequences on sperm quality (motility, count and morphology).…”

Reactive oxygen species as mediators of sperm capacitation and pathological damage

“…The significantly elevated levels of homocysteine observed in the spermatozoa of patients who exhibit severe disruptions to their semen protein-including asthenozoospermia, asthenoteratozoospermia, and oligoastheno teratozoospermia (Kralikova et al, 2017)-could reflect the ability of homocysteine to create a state of oxidative stress following conversion to thiolactone and subsequent activation of mitochondrial ROS generation. Similarly, the recent observation that Stattic V, an inhibitor of Signal transducer and activator of transcription 3, will trigger mitochondrial ROS in mammalian spermatozoa(Lachance, Goupil, Tremblay, & Leclerc, 2016) is probably a reflection of this compound's ability to form adducts with proteins (potentially in the electron transport chain) via thiol-mediated Michael addition reactions. The fact that N-acetyl-L-cysteine inhibited the ROSinducing properties of Stattic V would be in keeping with such a thiol-adduct formation mechanism(Lachance et al, 2016).…”

“…Similarly, the recent observation that Stattic V, an inhibitor of Signal transducer and activator of transcription 3, will trigger mitochondrial ROS in mammalian spermatozoa(Lachance, Goupil, Tremblay, & Leclerc, 2016) is probably a reflection of this compound's ability to form adducts with proteins (potentially in the electron transport chain) via thiol-mediated Michael addition reactions. The fact that N-acetyl-L-cysteine inhibited the ROSinducing properties of Stattic V would be in keeping with such a thiol-adduct formation mechanism(Lachance et al, 2016).…”