In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells, we showed ∼70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.

Seminal oxidative stress occurs when there is an increased production of reactive oxygen species (ROS) and/or a decrease of antioxidant activity, promoting impaired sperm function. Peroxiredoxins (PRDX) are abundant in human semen and are important antioxidant enzymes, which act as ROS scavengers and modulators in ROS-dependent signaling. Our aim was to determine whether the levels of PRDX1 and PRDX6 and their oxidation on thiol groups are associated with a decrease in sperm motility and DNA integrity. We evaluated the sperm and seminal PRDX level in men (13 healthy controls, 15 men with clinical varicocele, and 17 men with idiopathic infertility). We assessed conventional semen parameters, sperm DNA integrity (by the sperm chromatin structure assay), lipid peroxidation in seminal plasma and spermatozoa (by the thiobarbituric acid reactive substances assay), and the amount and thiol oxidation of PRDX1 and PRDX6 (by immunoblotting). PRDXs were affected in seminal plasma (lower amounts) and in sperm samples (lower amounts and higher levels of thiol oxidation) characterized by lower sperm motility, higher lipid peroxidation, and sperm DNA damage. The thioloxidation ratio of PRDXs (thiol-oxidized PRDX/total PRDX) correlated negatively with sperm motility (total and progressive) and positively with sperm DNA damage and sperm lipid peroxidation. In conclusion, because of the lower amount of total PRDX1 and PRDX6 and the high thiol oxidation of these PRDXs, very little (less than 20%) protection due to PRDXs remains, and this is associated with impaired sperm function and poor DNA integrity and suggests an important role of PRDXs in the protection of human spermatozoa against oxidative stress.

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