Mitochondrial uncouplers impair human sperm motility without altering ATP content

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. Significance Statement Development of novel contraceptives is critical, since half of all pregnancies are still unplanned, even in developed countries. This high unplanned pregnancy rate contributes to a wide variety of social, environmental, and ecological problems. Impairing human sperm is a way to develop male and unisex contraceptives, but much remains unknown about these unique cells. Here we settle a long-running debate about human sperm metabolism, finding that human sperm can maintain their ATP levels without mitochondrial oxidative phosphorylation. This finding will help focus future contraceptive development efforts. We also identify the potential use of an FDA-approved compound (Niclosamide) as a motility-impairing ingredient in spermicides and correct the misunderstood subcellular localization of an existing contraceptive target, Adenosine Nucleotide Translocator 4. ### Competing Interest Statement W.M.S., P.V.L, E.T., A.M.B., and Y.K are co-inventors on a patent related to this work, which UC Berkeley has licensed to YourChoice Therapeutics, for which P.V.L is a co-founder, and Y.K. is a shareholder. The other authors declare no competing interests. * ANT : Adenine nucleotide translocase ALH : Amplitude of lateral head displacement ATP : Adenosine triphosphate AUC : Area under the curve BAM15 : (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine ) BAPTA : (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) BSA : Bovine serum albumin CATR : Carboxyatractyloside COXIV : Cytochrome c oxidase subunit 4 isoform 1, mitochondrial DNP : 2, 4, Dinitrophenol EGTA : (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) FCCP : Trifluoromethoxyphenylhydrazone FDA : Food and Drug Administration HEPES : (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HRP : Horseradish peroxidase HS : High Saline HTF : Human Tubal Fluid ICC : Immunocytochemistry IMM : Inner mitochondrial membrane KSOM : Potassium-supplemented simplex optimized medium LIN : Linearity MES : 2-(N-morpholino)ethanesulfonate. MMP : Mitochondrial membrane potential NEN : Niclosamide Ethanolamine NMDG : N-methyl-D-glucamine PI : propidium iodide PMT : Photomultiplier tube PVDF : polyvinylidene difluoride STORM : Stochastic optical reconstruction microscopy Tris : tris(hydroxymethyl)aminomethane VCL : Curvilinear velocity