Abstract
The sperm plasma membrane is a sensitive target to oxidative stress. The most representative reactive oxygen species (ROS) scavengers in the genital tract, hypotaurine and glutathione, require, for their synthesis, cysteine whose availability is associated with the 1-carbon cycle (1-CC). Human, bovine and ascidian spermatozoa were incubated with compounds supporting the 1-CC (Vitamin B6, Methylcobalamin, 5 Methyl Tetrahydrofolate, Zinc Bisglycinate and N-acetyl-cysteine) (TRT) and compared to the effects induced solely by N-acetyl-cysteine (NAC). In control groups (CNTRL), spermatozoa were incubated with medium alone. After 90 and 180 minutes of incubation, the mitochondrial membrane potential (ΔΨM) in TRT and NAC was significantly (P < 0.01) higher than in CNTRL. At H2DCFDA evaluation, ROS production differed between species whereas, at 2-OH Ethidium, it significantly decreased in bovine TRT group. Intracellular pH (pHi) did not significantly vary in relation to treatment. In ascidian spermatozoa, the NAC supplementation decreased external pH, which in turn brought to a pHi lowering. Buffering seawater with NaHCO3 reversed the beneficial effects of N-acetyl-cysteine supplementation. In conclusion, both fully supporting the 1-CC and treatment with N-acetyl-cysteine alone improved kinetics, ΔΨM and ROS production in mammalian sperm demonstrating for the first time the direct in vitro effects of these compounds on sperm functionality.
Highlights
The spermatozoon is a highly differentiated cell with a compacted nucleus, scarce cytoplasm, mitochondria and usually a flagellum
The beneficial effects observed in TRT and N-acetyl cysteine (NAC) groups were not sufficient to effectively counteract the natural decline of motility which was inferior to control groups (CNTRL) at time 0 even after 90 min incubation (48.2 ± 2.3% and 51.2 ± 2.5% vs. 70.1 ± 1.4%; P < 0.01)
The 1-carbon cycle (1-CC) is an important target for maintaining sperm quality as it is involved in methylation and generation of three major antioxidants: hypotaurine, glutathione and Coenzyme Q1023,24
Summary
The spermatozoon is a highly differentiated cell with a compacted nucleus, scarce cytoplasm, mitochondria and usually a flagellum. The high mitochondrial activity of spermatozoa together with the small amount of cytoplasm and a high concentration of polyunsaturated fatty acids in plasma membrane makes these cells extremely sensitive to oxidative damage, which may affect DNA, proteins and membrane integrity In the latter case, motility[8], adhesion and fusion during fertilization process and the acrosome reaction may be strongly impaired[9,10]. The effects of five compounds involved in the 1-CC, i.e., Vitamins B6 and methyl B12, 5 methyl folate, zinc and N-acetyl cysteine (NAC) on different sperm parameters, such as mitochondrial activity, lipid peroxidation, ROS production and intracytoplasmic pH, were evaluated in human, bovine and the ascidian C. robusta. This treatment was compared to the impact of NAC alone, since in preliminary trials (Supplemental Fig. S2) this compound showed a predominant activity compared to that of the other individually evaluated enhancers
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