Single-cell Motility Analysis of Tethered Human Spermatozoa

Biology of Reproduction

Petersen

Unger

et al. 2023

In human spermatozoa, the electrochemical potentials across the mitochondrial and plasma membranes are related to sperm functionality and fertility, but the exact role of each potential has yet to be clarified. Impairing sperm mitochondrial function has been considered as an approach to creating male or unisex contraceptives, but it has yet to be shown whether this approach would ultimately block the ability of sperm to reach or fertilize an egg. To investigate whether the mitochondrial and plasma membrane potentials are necessary for sperm fertility, human sperm were treated with two small-molecule mitochondrial uncouplers (Niclosamide Ethanolamine and BAM15) that depolarize membranes by inducing passive proton flow, and evaluated the effects on a variety of sperm physiological processes. BAM15 specifically uncoupled human sperm mitochondria while Niclosamide Ethanolamine induced proton current in the plasma membrane in addition to depolarizing the mitochondria. Additionally, both compounds significantly decreased sperm progressive motility with Niclosamide Ethanolamine having a more robust effect. However, these uncouplers did not reduce sperm ATP content or impair other physiological processes, suggesting that human sperm can rely on glycolysis for ATP production if mitochondria are impaired. Thus, systemically delivered contraceptives that target sperm mitochondria to reduce their ATP production would likely need to be paired with sperm-specific glycolysis inhibitors. However, since Niclosamide Ethanolamine impairs sperm motility through an ATP-independent mechanism, and Niclosamide is FDA-approved and not absorbed through mucosal membranes, it could be a useful ingredient in on-demand, vaginally-applied contraceptives.

Section: Methodsmentioning

confidence: 99%

Mitochondrial uncouplers impair human sperm motility without altering ATP content

Biology of Reproduction

Petersen

Unger

et al. 2023

“…S4 and S5 were approved by the Committee on Human Research at the University of California, Berkeley, IRB protocol 2013-06-5395. Purified human sperm (5) were lysed in a buffer of 150 mM NaCl, 50 mM Tris pH 7.4, 1% Triton X-100, 0.5% Sodium deoxylcholate, 0.1% SDS, 1 mM EDTA, 10% (v/v) glycerol, and Halt protease inhibitors (ThermoFisher). The protein concentrations of the whole cell lysates were estimated by a Bradford assay (BioRad) using bovine serum albumin as a standard.…”

Section: Si Materials and Methodsmentioning

confidence: 99%

Human sperm TMEM95 binds eggs and facilitates membrane fusion

Tang

et al. 2022

Preprint

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino-acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion.

“…Human sperm cells were collected by masturbation from healthy donors and visually inspected for normal morphology and motility before use. Spermatozoa were isolated by the swim-up procedure in HTF or HS solution as previously described (46) and then concentrated by 5 minute centrifugation at ≤500g and supernatant removal.…”

Section: Sperm Preparationmentioning

confidence: 99%

Mitochondrial uncouplers impair human sperm motility without altering ATP content

Petersen

Unger

et al. 2022

Preprint

Sperm motility is necessary for successful fertilization, but there remains controversy about whether human sperm motility is primarily powered by glycolysis or oxidative phosphorylation. To evaluate the plausibility of reducing human sperm mitochondrial ATP production as an avenue for contraceptive development, we treated human sperm with small-molecule mitochondrial uncouplers, which reduce mitochondrial membrane potential by inducing passive proton flow, and evaluated the effects on a variety of physiological processes that are critical for fertilization. We also sought to clarify the subcellular localization of Adenosine Nucleotide Translocator 4 (ANT4), a gamete-specific protein that has been suggested as a contraceptive target. We determined that ANT4 is mitochondrially localized, that induced mitochondrial uncoupling can be partially mediated by the ANT family, and that two uncouplers, Niclosamide Ethanolamine and BAM15, significantly decreased sperm progressive motility. However, these uncouplers did not reduce sperm ATP content or impair other physiological processes, implying that human sperm can rely on glycolysis for ATP production in the absence of functional mitochondria. Thus, since certain mitochondrial uncouplers impair motility through ATP-independent mechanisms, they could be useful ingredients in on-demand, vaginally-applied contraceptives. However, systemically delivered contraceptives that target sperm mitochondria to reduce their ATP production would need to be paired with sperm-specific glycolysis inhibitors.