Sperm Toolbox-A selection of small molecules to study human spermatozoa

Abstract

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying.

Fig 1. Design and assay of the Sperm Toolbox.

(A) The Sperm Toolbox is a collection of 83 small molecules with published, observed or inferred effect on multiple biological processes related to the biogenesis or function of spermatozoa. Human sperm cells have been tested in various assays, which allows comparison of compounds under the same experimental conditions. (B) Summary of the STB compound compositions. (C) Summary of annotated (i.e. published, observed, inferred) sperm function of Toolbox compounds. (D) Coarse classification of STB target classes. (E) StringDB Protein-Protein interaction networks (Full network, confidence 0.7) of putative targets of STB compounds. Protein targets have been inferred from the repurposing hub. Network has been clustered using MCL clustering in Cytoscape. (F) Sperm related terms (GeneOntology, Reactome) within the network shown in (E). Marker size indicates false discovery rate (FDR). (G) Enriched terms within the biggest subclusters in (E). Redundant terms have been filtered using Cytoscape (cutoff 0.5). Color matches subcluster color in (E).

Fig 2. Example assay data from STB compounds.

(A) Motility assay example images. Tracked sperm cells treated with DMSO (top row), LRRK2-IN-1 (middle row) or Tafenonquine (bottom row). First (0 sec) and last frame (0.5 sec) of time-lapse movies are shown. Non-capacitating conditions (3h incubation with compound) are shown next to capacitating conditions (30 min incubation with compound). Compound concentration was 10 uM for all shown conditions. Colors in the non-capacitating assay indicate immotile sperm (pink), non-progressively motile sperm (lavender) or progressively motile sperm (teal). Colors in the capacitation assay indicate hyperactive sperm (green) or non-hyperactive sperm (blue). Scale bars 50 um. (B) Acrosome assay examples. Histogram depicting number of events and shift in intensity in the PNA acrosome channel (AR+ population). Acrosome assay protocol 1 (3h incubation with compound, no agonist addition; left panel) and acrosome assay protocol 2 (3h incubation with compound, 1h with agonist; right panel). Comparing DMSO (top row), LRRK2-IN-1 (middle row) and Tafenoquine (bottom row). Compound concentration was 10 mM for all shown conditions. Dashed line indicates gate (AR+ population).

Fig 3. Visualization of assay results on STB compounds.

(A) Heatmap showing standardized results (z-score) for every Toolbox compound screened at 30 μM in selected assays: Acrosome 2 (3h with compound then induction with acrosome reaction using an agonist mix), Motility Hyperactive (30 min capacitation then 30 min incubation with compound), Motility Progressive (3h incubation with compounds under non-capacitating conditions), Motility Total (3h incubation with compound under non-capacitating conditions)), Solubility (aqueous solubility of compounds after 24h incubation), Sperm viability (3h with compound then propidium iodide assay), Toxicity HeLa (24h compound incubation then cell painting assay), Toxicity HepG2 (70h incubation with compound then resaruzin based assay). Heatmap color indicates decrease (blue) or increase (red). (B) UMAP plot showing groups of compounds with similar assay profile. Color indicates annotated sperm function. (C) Structures and zoom-in of three compounds with related structures described to modulate LRRK2 kinase but appear to have different effects on sperm function.