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Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm
In This Article
Summary
Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.
Abstract
Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.
Introduction
The purpose of the two assays described here is to examine effects on Ca2+-signaling and acrosome reaction in human sperm, as has been shown for multiple compounds in several publications employing these assays1,2,3,4,5,6,7. Ca2+-signaling and the acrosome reaction are both vital to normal human sperm cell function and male fertility.
The overall goal of a human sperm cell is to fertilize ....
Protocol
The collection and analysis of human semen samples in the protocols follows the guidelines of the Research Ethics Committee of the Capital Region of Denmark. All semen samples have been obtained after informed consent from volunteer donors. After delivery, the samples were fully anonymized. For their inconvenience each donor received a fee of 500 DKK (about $75 US dollars) per sample. The samples were analyzed on the day of delivery and then destroyed immediately after the laboratory experiments.
Representative Results
Representative results from an experiment testing the effect of 4 compounds (A, B, C, and D) together with a positive (progesterone) and negative (buffer) control on [Ca2+]i in human sperm using the medium-throughput Ca2+-signaling assay can be seen in Figure 4a. In Figure 4b, a dose response curve of progesterone is shown, which was derived from peak ΔF/F0 (%) data induced by seri.......
Discussion
The medium throughput Ca2+ signaling assay is based on measurements of fluorescence from single microwells each containing about 250,000 sperm cells. The captured signal is averaged from all individual sperm cells in the well. The assay thus provides no spatial information about where specifically in the sperm cell [Ca2+]i is changed, in how large a proportion of the sperm cells a change in [Ca2+]i takes place, or how heterogeneous the response is between the individ.......
Acknowledgements
The authors would like to acknowledge the lab of Timo Strünker for supervision of AR and DLE during their stays at his lab. Furthermore, we would like to thank our colleagues at the Department of Growth and Reproduction, Copenhagen University Hospital, Rigshospitalet for their assistance with setting up these two assays. This project was supported by the Danish Environmental Protection Agency as a project under Centre on Endocrine Disrupters and by grants from the Innovation Fund Denmark (grant numbers 005-2010-3 and 14-2013-4).
....Materials
| Name | Company | Catalog Number | Comments |
| 0.2 µm pore filter | Thermo Fisher Scientific, USA | 296-4545 | |
| 1 L measuring cylinder | Thermo Fisher Scientific, USA | 3662-1000 | |
| 1,4 and 2 mL plastic tubes | Eppendorf, Germany | 30120086 and 0030120094 | |
| 12-channel pipette | Eppendorf, Germany | 4861000813 | |
| 384 multi-well plates | Greiner Bio-One, Germany | 781096 | |
| 15 and 50 mL platic tubes | Eppendorf, Germany | 0030122151 and 30122178 | |
| A2-slide | ChemoMetec, Denmark | 942-0001 | |
| Automatic repeater pipette | Eppendorf, Germany | 4987000010 | |
| CaCl2 | |||
| Centrifuge | |||
| Clean wide-mouthed plastic container for semen sample | |||
| Dimethyl sulfoxide (DMSO) | |||
| FITC-coupled lectin of Pisum sativum (FITC-PSA) | Sigma-Aldrich, Germany | L0770 | |
| Fluo-4 AM | Thermo Fisher Scientific, USA | F14201 | |
| FLUOstar OMEGA fluorescence microplate reader | BMG Labtech, Germany | ||
| Glucose anhydrous | |||
| HEPES | |||
| Hoechst-33342 | ChemoMetec, Denmark | 910-3015 | |
| Human serum albumin (HSA) | Irvine Scientific | 9988 | For addition to HTF+ |
| Immobilizing solution containing 0.6 M NaHCO3 and 0.37% (v/v) formaldehyde in distilled water | |||
| Incubator | |||
| KCl | |||
| KH2PO4 | |||
| Magnetic stirrer | |||
| MgSO4 | |||
| Na-Lactate | |||
| NaCl | |||
| NaHCO3 | |||
| NC-3000 image cytometer | ChemoMetec, Denmark | 970-3003 | |
| Pipettes and piptting tips | |||
| propidium iodide (PI) | ChemoMetec, Denmark | 910-3002 | |
| Purified water | |||
| Rack for placing 50 mL plastic tubes in 45° angle | |||
| S100 buffer | ChemoMetec, Denmark | 910-0101 | |
| SP1-cassette | ChemoMetec, Denmark | 941-0006 | |
| Volumetric flasks of appropriate sizes | |||
| Vortexer |
References
- Schiffer, C., et al. Direct action of endocrine disrupting chemicals on human sperm. EMBO reports. , (2014).
- Rehfeld, A., Dissing, S., Skakkebæk, N. E. Chemical UV Filters Mimic the Effect of Proges....
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