Hydrogen sulphide (H 2 S) is involved in the physiology and pathophysiology of different cell types, but little is known about its role in sperm cells. Because of its reducing properties, we hypothesise that H 2 S protects spermatozoa against the deleterious effects of oxidative stress, a condition that is common to several male fertility disorders. This study aimed i) to determine the total antioxidant capacities of Na 2 S and GYY4137, which are fast-and slow-releasing H 2 S donors, respectively, and ii) to test whether H 2 S donors are able to protect spermatozoa against oxidative stress. We found that Na 2 S and GYY4137 show different antioxidant properties, with the total antioxidant capacity of Na 2 S being mostly unstable and even undetectable at 150 µM. Moreover, both H 2 S donors preserve sperm motility and reduce acrosome loss, although the effects were both dose and donor dependent. Within the range of concentrations tested (3-300 µM), GYY4137 showed positive effects on sperm motility, whereas Na 2 S was beneficial at the lowest concentration but detrimental at the highest. Our findings show that Na 2 S and GYY4137 have different antioxidant properties and suggest that both H 2 S donors might be used as in vitro therapeutic agents against oxidative stress in sperm cells, although the optimal therapeutic range differs between the compounds. Hydrogen sulphide (H 2 S) is the most recently discovered gaseous molecule that participates in a variety of biological functions, as do nitric oxide (NO) and carbon monoxide (CO). In mammals, H 2 S can be synthesised by enzymatic or non-enzymatic pathways 1. Overall, it seems likely that most of the H 2 S produced within an organism is generated by the H 2 S-synthesising enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulphurtransferase (3-MST), with the latter coupled with cysteine aminotransferase (CAT) 2. In the male reproductive system, the expression of H 2 S-generating enzymes has been reported in the testis 3,4 , epididymis 5 , penile corpus cavernosum 6 , and spermatozoa 7,8 , which strongly suggests that this gasotransmitter is involved in sperm physiology to some extent. In a recent study, Wang et al. found that asthenospermic men show reduced levels of H 2 S in their seminal plasma and that exogenous H 2 S supplementation improves their sperm motility 8. In contrast, in boar spermatozoa, H 2 S exerts no or negative effects on sperm motility, viability, and mitochondrial membrane potential 9. With both positive and negative effects documented, there is still controversy concerning the role of H 2 S in sperm cells. This apparent discrepancy might, at least partly, be a result of H 2 S dose-and donor-dependent effects 10. According to their chemical structure and source, H 2 S donors include inorganic salts and derivatives of phosphorodithioate, garlic extracts, thioaminoacids, and anti-inflammatory drugs 11. On the basis of their release mechanism, H 2 S donors can be classified in two categories: slow-and fast-re...

Aminoguanidine is a selective inhibitor of the inducible nitric oxide synthase (iNOS) and a scavenger of reactive oxygen species (ROS). Numerous studies have shown the antioxidant properties of aminoguanidine in several cell lines, but the in vitro effects of this compound on spermatozoa under oxidative stress are unknown. In this study, we tested the hypothesis that aminoguanidine may protect against the detrimental effects of oxidative stress in boar spermatozoa. For this purpose, sperm samples were incubated with a ROS generating system (Fe2+/ascorbate) with or without aminoguanidine supplementation (10, 1, and 0.1 mM). Our results show that aminoguanidine has powerful antioxidant capacity and protects boar spermatozoa against the deleterious effects of oxidative stress. After 2 h and 3.5 h of sperm incubation, the samples treated with aminoguanidine showed a significant increase in sperm velocity, plasma membrane and acrosome integrity together with a reduced lipid peroxidation in comparison with control samples (p < 0.001). Interestingly, except for the levels of malondialdehyde, the samples treated with 1 mM aminoguanidine did not differ or showed better performance than control samples without Fe2+/ascorbate. The results from this study provide new insights into the application of aminoguanidine as an in vitro therapeutic agent against the detrimental effects of oxidative stress in semen samples.

After being historically considered as noxious agents, nitric oxide (NO) and hydrogen sulfide (H2S) are now listed as gasotransmitters, gaseous molecules that play a key role in a variety of cellular functions. Both NO and H2S are endogenously produced, enzymatically or non-enzymatically, and interact with each other in a range of cells and tissues. In spite of the great advances achieved in recent decades in other biological systems, knowledge about H2S function and interactions with NO in sperm biology is in its infancy. Here, we aim to provide an update on the importance of these molecules in the physiology of the male gamete. Special emphasis is given to the most recent advances in the metabolism, mechanisms of action, and effects (both physiological and pathophysiological) of these gasotransmitters. This manuscript also illustrates the physiological implications of NO and H2S observed in other cell types, which might be important for sperm function. The relevance of these gasotransmitters to several signaling pathways within sperm cells highlights their potential use for the improvement and successful application of assisted reproductive technologies.

In recent decades, an increasing number of ethnopharmacological studies have been dedicated to medicinal plants from South African fynbos. Among these plants, honeybush (Cyclopia spp.) has become a popular tea, mainly due to its healthy properties and caffeine-free status. The antioxidant, antimutagenic, and antimicrobial properties of this plant have been reported in several cell types, but its effects on reproductive function are still unknown. Here, we assessed the effects of honeybush (Cyclopia intermedia) on boar sperm parameters under induced oxidative stress (Fe2+/ascorbate) and during five days of semen storage at 17 °C without oxidative stress. In both experiments, four concentrations (200, 50, 12.5, and 3.125 µg/mL) of fermented honeybush were tested. Our results show that honeybush enhances sperm parameters, and no toxic effects were observed at any of the tested extract concentrations. Interestingly, honeybush (12.5 µg/mL) improved the sperm motility and kinetic parameters, preserved the plasma membrane integrity, and reduced the lipid peroxidation in the samples exposed to Fe2+/ascorbate (p < 0.05). In the stored samples, positive effects of honeybush on sperm parameters (motility, kinetics, acrosome, and mitochondria) were observed from 48 h until 120 h of semen storage (p < 0.05). Our results clearly show the protective effects of honeybush on sperm samples, thus promoting its use as a natural source of antioxidants for boar semen.

Background Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. Results Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. Conclusions The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.