Sperm Cell Painting: A Mechanism Driven Approach for Drug Discovery in Human Spermatozoa

We have adapted the cell painting assay developed by Carpenter and colleagues on cultured U2OS cells to human spermatozoa. In Sperm Cell Painting (SCP) we assemble an image-based quantitative fingerprint of the functional state of sperm. We use this assay to gain insight into the mechanism of action of compounds that modify sperm function and as a platform for contraceptive discovery.


MAIN TEXT
Despite recent advances in the tools and technologies used to study human reproductive health, and a resurgence in interest around nonhormonal contraception, there remains a lack of knowledge and understanding of the fundamental biology underpinning human sperm function.This gap in understanding limits the diagnosis and treatment of infertility and slows the development of effective new contraceptive options.
To begin to address these gaps in understanding, we have recently developed a highthroughput phenotypic screening platform in human sperm and used it to screen for compounds that either both decrease [1] and enhance [2] sperm motility.This platform presents opportunities for target-agnostic screening which can be adapted for screening of specific sperm functions (reviewed in Johnston et al., 2021 [3] ). Target agnostic phenotypic assays provide a powerful, unbiased approach to drug discovery, but require substantial downstream follow-up of any hits to identify targets and assess the opportunity for further compound development.Each assay is also limited to the examination of individual sperm functions such as motility or acrosome reaction.
Cell Painting is an image-based, high-content method using multiplexed fluorescent dyes for cytological profiling [4].Images of cells can be converted to quantitative feature fingerprints and be used in screening assays and to classify [5,6] the mechanism of action (MOA) and/or toxicity of drugs and drug candidates, genetic perturbations [5,6] (Figure 1A) and is particularly powerful when combined with curated libraries of references compounds with known MOA, or known phenotypic effect, such as the Sperm Toolbox [7].
In this study, our aim was to adapt this powerful, high-throughput approach for use with human spermatozoa.Unlike the adherent, somatic cells that have previously been categorised by cell painting [4,8], spermatozoa are not only motile, but lack some of the cellular components/machinery of somatic cells such as the endoplasmic reticulum.Therefore, significant adaptations are required to utilise the techniques with human sperm cells (see Methods).
A major goal of this work was to develop a phenotypic screening platform for the discovery of a female-controlled contraceptive that targets spermatozoa. .After ejaculation, spermatozoa experience different physiological states as they travel within the female reproductive tract.In response, they undergo a process known as capacitation, which involves destabilization of the acrosome membrane and changes in the tail that promote motility and fertilisation competence (reviewed in Puga Molina et al., 2018 [9]).Although the physiological and behavioural changes associated with capacitation have been studied in detail, the molecular basis for capacitation is not well-characterized.For contraceptive development, it is therefore an ideal functional target, but identification of molecular targets will require a screening strategy that provides as much mechanistic detail as possible.In addition, as the process of capacitation is highly species-specific, we focussed on assays that employed living human sperm to ensure compound hits would have effects in humans.
For these reasons, we have designed an assay strategy that supports capacitation in human sperm, and designed compound incubation protocols (Figure 1B) that model short (1 hour; Protocol A) compound exposure on capacitated cells and long exposure (3 hours; Protocol B) on cells during the capacitation process, allowing us to model the effects of compound exposure at different points during the journey of sperm through the female reproductive tract.
We incubated compounds from several small, well annotated libraries with living spermatozoa and then processed cells for Cell Painting (Figure 2A).We used CellProfiler to identify cells and segment nuclei, acrosome membrane and the mitochondria and calculate image-based features.Feature normalisation and scaling was performed as described in Bray et al (2016;[4]).We filtered compounds using an induction score (Figure 2B), and selected compounds for further analysis where >15% of features were >= 2σ deviation from DMSO (Figure 2C), which produced 140 and 146 compounds using Protocol A and B respectively (Figure 2D), of which 56 were observed in both exposure protocols (Figure 2E).The pattern of z-scores in the heatmaps of the two are visibly different (Figure 2 F & G), indicating differential responses to compounds by cells subject to different exposure protocols.Furthermore, we observed that the annotated mechanism of actions of these compounds are similar, but different with different compound exposure protocols (Figure 2H).Both exposure protocols share a majority of mechanisms of action, however Protocol A detects more estrogen and dopamine receptor related compounds, while Protocol B detects sodium channel, serotonin receptor and cyclooxygenase related compounds.This highlights the potential for information gained from this approach to define pathways for further screening or for target deconvolution, and the importance of selecting screening conditions that reflect the relevant physiological state of the target cell.
To better understand similarities between compounds and exposure protocols, we calculated pairwise correlation distance and generated cluster labels using hierarchical clustering (Figure 3A).This shows that there are multiple mechanistic clusters sharing profile similarity.
In addition, we used UMAP dimensionality reduction to visualise groups of compounds with similar feature profiles to each other in the Sperm Cell Painting assay (Figure 3B).As with the heatmap, UMAP revealed that both incubation states generate similar overall patterns, but with visible differences (Figure 3C).This result confirms our hypothesis that different target engagement occurs in cells in different physiological states or with different incubation times.
These visualisations can also be used to map phenotypes in orthogonal sperm assays.
Compounds which either increase or decrease hyperactive motility in our capacitation assay (See online methods -Motility Assays) are dispersed throughout each of the clusters (Figure 3D).These effects are seen both in addition to, and independently from changes to progressive motility for individual compounds (Figure 3D).Similarly, cross-referencing these clusters with datasets from our original non-capacitated motility assay (See online methods -Motility Assays) reveals that hits from the motility and capacitation assay do not lie within any one cluster (Figure 3E).These results are consistent with the hypothesis that multiple molecular pathways are likely to be involved in the regulation of motility and capacitation [9].
One potential application of the Sperm Cell Painting assay is to look for compounds which affect the acrosome reaction (AR).We have used control compounds in our assays which artificially induce AR, such as A23187 a well characterized Ca2+ ionophore.A23187 is part of Cluster 2 (Figure 4A).This cluster contains an accumulation of compounds that have an annotated mechanism of action related to adrenergic, histaminergic, and serotonergic receptors (Figure 4B).Images of compounds with a similar feature profile as A23187 show that these compounds induce very high levels of typical acrosomal banding pattern staining, suggesting that they do induce acrosome reaction (Figure 4C).These data suggest that targets related to serotonin receptors, a receptor coupled GTPase, and at least one protein kinase are potential, previously unknown, targets for premature induction of AR and thus candidates for drug development programs.Combining Sperm Cell Painting with targetdirected assays will be a powerful approach for future contraceptive and fertility drug development.The induction of the acrosome reaction at high frequency also confirms that our assay are effectively supporting capacitation.
In summary, we have successfully transferred the well-established Cell Painting assay to a non-somatic and motile cell type.We observe differences related to the exposure Protocol used, highlighting the importance of screening in conditions which reflect the appropriate physiological state of the cell for the target application.We also observe mechanistic clusters of compounds sharing similar feature profiles, which presents an advantage for efficient drug discovery programmes.The ability to combine mechanistic and phenotypic approaches to screen and compare the data from the Sperm Cell Painting assay with that from other phenotypic assays has enabled us to create rich datasets that could prove invaluable in driving forward knowledge in male (in)fertility and drug-discovery.The use of well-defined and annotated libraries such as the Sperm toolbox [7] further increases the relevance of the information gained from screens to benchmark new assay and define areas of human sperm biology.

Assay ready plates
Assay-ready 384-well plates were prepared prior to the screen.Library compounds (10mM stocks in DMSO) were dispensed using an acoustic dispenser (Echo 555, Labcyte Inc) to the desired concentration in poly-D-lysine-coated plates (poly-D-lysine-coated PhenoPlate 384well; PerkinElmer; 6057500).The positions of each compound in the plate were allocated at random, and DMSO concentration was consistent between wells.
The Prestwick Chemical Library contains off patent drugs with known human bio-availability and safety profiles (https://www.prestwickchemical.com/screening-libraries/prestwickchemical-library/)and contained 1280 compounds at the time of purchase.
The CeMM library of unique drugs (CLOUD; https://enamine.net/compoundlibraries/bioactive-libraries/the-comprehensive-drug-collection-cloud)contained 262 compounds at the time of purchase.The library contains a set of small molecules representing the chemical space of FDA-approved drugs.
The SelleckChem library contains 219 cherry picked annotated bioactive compounds.
The Sperm Toolbox is a library of 85 small molecules assembled specifically for the study of human spermatozoa [7].

Compound exposure
Two compound exposure protocols were used in order to examine the effects of compound exposure on cells in different physiological states.For both cases, screening batches of cells were transferred to a robotic platform (HighRes Biosolutions Inc.) and maintained at 37°C.
Cells were dispensed into assay-ready 384-well plates at approximately 100,000 spermatozoa per well using a using a liquid handling system (MultiDrop Combi; ThermoFisher).Cells were then incubated in these assay plates, in the presence of compounds, for either 1 or 3 hours (Figure 1B).

Exposure Protocol A:
Prepared sperm cells were incubated in conditions which support capacitation (HTF) for three hours, prior to being dispensed into assay-ready 384-well plates containing compounds.The cells, which had been given time to undergo capacitation prior to addition to the plates, were then incubated with compounds within these plates for 1 hour prior to staining and fixation.
Exposure Protocol B: After a 30 minute rest period, prepared sperm cells were dispensed into assay-ready 384-well plates containing HTF and compounds.Cells were then incubated for 3 hours within the assay plate, and were therefore exposed to compounds during capacitation.

Staining protocol
Plates were stained with a combination of dyes targeting the nucleus, acrosome, and mitochondria of the sperm cells.Bioconductor package clusterprofiler to study gene ontology (GO) enrichments or potential protein targets.

Motility Assays
Compound induction profiles in the sperm cell painting assay were compared to the effects of compound on sperm motility using two forms of high-throughput motility assay.
Progressive motility was measured using the high-throughput assay previously published in Gruber et al., 2020 and 2022 [1,2] in non-capacitating conditions and Non-capacitation media.
The effect of compound exposure on the hyperactivated motility of sperm cells was measured using a modification of this assay platform that we have termed the capacitation assay.
Prepared cells were incubated in HTF medium for 30 minutes at 37°C and 5% CO2 prior to dispensing into an un-coated assay ready plate.Plates were further incubated for 30 minutes at 37°C and 5% CO2.After 30 minutes compound exposure, plates were imaged using the CV7000 (Yokogawa) and cell motility tracked and classified as in Gruber et al., 2020 [1].

Figure 3 166Figure 3 Figure 4 169Figure 4
Figure 3 Fixation and adherenceStocks of 4% PFA were prepared from PFA powder (Sigma 441244) in MQ-H2O and pH adjusted to pH 7 with addition of NaOH.An equal volume (20µL) of 4% PFA was dispensed by TEMPEST to each well, for a final concentration of 2% PFA, and incubated for a further 10 minutes at room temperature.To adhere the now-fixed cells to the poly-D-lysine-coated well bottom, each plate was centrifuged at 300g for 5 minutes in each of two orientations.The microplates were then washed gently using an automated Bio Tek 405 LS Washer (Agilent)ImagingImages were captured on an ImageXpress Micro XLS epifluorescence microscope (Molecular Devices) or CV7000 (Yokogawa) in three fluorescent channels.