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.
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.
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 the egg. To be able to successfully and naturally fertilize the egg, the functions of the sperm cell must be regulated tightly during the journey of the sperm cell through the female reproductive tract8,9. Many of the sperm cell functions are regulated via the intracellular Ca2+-concentration [Ca2+]i (e.g., sperm motility, chemotaxis, and acrosome reaction)10. Also, a maturation process called capacitation, which renders the sperm cell capable of fertilizing the egg, is partly regulated by [Ca2+]i10. Ca2+-extruding Ca2+-ATPase pumps11 maintain an approximately 20.000 fold Ca2+-gradient over the human sperm cell membrane, with a resting [Ca2+]i of 50-100 nM. If Ca2+ is allowed to cross the cell membrane (e.g., through the opening of Ca2+-channels), a sizeable influx of Ca2+ occurs, giving rise to an elevation of [Ca2+]i. However, the sperm cell also carries intracellular Ca2+-stores, which can release Ca2+ and, therefore, also give rise an elevation of [Ca2+]i12. Interestingly, all channel-mediated Ca2+-influx in human sperm cells has so far been found to occur via CatSper (Cationic channel of Sperm), which is only expressed in sperm cells11. In human sperm cells, CatSper is activated by the endogenous ligands progesterone and prostaglandins through distinct ligand binding sites13,14,15, leading to a rapid Ca2+-influx into the sperm cell. Two main sources near the egg provide high levels of these endogenous ligands. One is the follicular fluid that contains high levels of progesterone16. The follicular fluid is released from mature follicles together with the egg at ovulation and mixes with the fluid within the oviducts17. The other main source is the cumulus cells that surround the egg and release high levels of progesterone and prostaglandins. The progesterone-induced Ca2+-influx in the sperm cells has been shown to mediate chemotaxis towards the egg9,18, control sperm motility19,20 and stimulate the acrosome reaction21. Triggering of these individual [Ca2+]i-regulated sperm functions in the correct order and at the correct time is crucial for fertilization of the egg8. In line with this, a suboptimal progesterone-induced Ca2+-influx has been found to be associated with reduced male fertility22,23,24,25,26,27,28,29 and functional CatSper is essential for male fertility26,30,31,32,33,34,35,36.
As the sperm cells reach the egg, a sequence of events must take place for fertilization to occur: 1) The sperm cells must penetrate the surrounding cumulus cell layer, 2) bind to the zona pellucida, 3) exocytose the acrosomal content, the so-called acrosome reaction37, 4) penetrate the zona pellucida, and 5) fuse with the egg membrane to complete fertilization38. To be able to go through these steps and fertilize the egg, the sperm cell must first undergo capacitation11, which begins as the sperm cells leave the seminal fluid containing "decapacitating" factors39 and swim into the fluids of the female reproductive tract with high levels of bicarbonate and albumin37. Capacitation renders the sperm cells able to undergo hyperactivation, a form of motility with a vigorous beating of the flagellum, and acrosome reaction37. Hyperactivated motility is required for penetration of the zona pellucida40, and the acrosome contains various hydrolytic enzymes that aid this penetration process41. Additionally, the acrosome reaction renders the sperm cells capable of fusing with the egg by exposing specific membrane proteins on the sperm surface necessary for sperm-egg fusion42. Consequently, the ability to undergo hyperactivation and acrosome reaction are both required for successful fertilization of the egg40,42. Contrary to what has been seen for mouse sperm cells43,44,45, only human sperm cells that are acrosome-intact can bind to the zona pellucida46. When the human sperm cells are bound to the zona pellucida they have to undergo the acrosome reaction both to penetrate the zona pellucida41 and to expose specific membrane proteins that are needed for the fusion with the egg38. The timing of the acrosome reaction in human sperm is thus critical for fertilization to occur.
As described above, Ca2+-signaling is vital for normal sperm cell function8, and it is, therefore, of great interest to be able to screen large numbers of compounds for effects on Ca2+-signaling in human sperm cells. Similarly, as only human sperm cells that undergo acrosome reaction at the right time and place can penetrate the zona pellucida and fertilize the egg46,47, it is also of great interest to be able to test compounds for their ability to affect the acrosome reaction in human sperm. To this end, two medium-throughput screening assays are described: 1) an assay for effects on Ca2+-signaling in human sperm cells, and 2) an assay for the ability to induce acrosome reaction in human sperm cells.
Assay 1 is a medium-throughput Ca2+-signaling assay. This fluorescence plate reader-based technique monitors changes in fluorescence as a function of time simultaneously in multiple wells. The Ca2+-sensitive fluorescent dye, Fluo-4 has a Kd for Ca2+≈ 335 nM and is cell-permeant in the AM (acetoxymethyl) ester form. Using Fluo-4, it is possible to measure changes in [Ca2+]i over time and after the addition of compounds of interest to the sperm cells. The assay was developed by the lab of Timo Strünker in 201113 and has since been used in several studies to screen compounds for effects on Ca2+-signaling in human sperm1,2,3,4,5. Also a similar method has been used to screen multiple drug candidates48. In addition, this assay is also useful for assessing the pharmacological mode of action1,2,3,4,5, dose-response curves1,2,3,4,5, competitive inhibition1,2, additivity1,2, and synergism3 of compounds of interest.
Assay 2 is a medium-throughput acrosome reaction assay. This image cytometer-based technique measures the amount of viable acrosome reacted sperm cells in a sample, using three fluorescent dyes: propidium iodide (PI), FITC-coupled lectin of Pisum sativum (FITC-PSA), and Hoechst-33342. The assay was modified from a similar flow cytometry-based method by Zoppino et al.49 and has been used in several studies6,7. As for the Ca2+-signaling assay, this acrosome reaction assay could also be used to assess dose-response curves, inhibition, additivity, and synergism of compounds of interest.
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.
NOTE: The medium throughput Ca2+-signaling assay is described in steps 4-5 and the medium throughput acrosome reaction assay in steps 6-7. The protocol to prepare human tubal fluid (HTF+) medium is described in step 1 and the purification of sperm cells for the assays is described in steps 2-3.
1. Preparation of Human Tubal Fluid (HTF+) Medium
NOTE: Use volumetric flasks of appropriate sizes, a 1 L measuring cylinder, a magnetic stirrer, salts as listed in Table 1 and Table 2, purified water, a 0.2 µm pore filter, and 50 mL plastic tubes.
2. Purification of Motile Sperm Cells via Swim-up (Figure 1)
NOTE: Use a clean wide-mouthed plastic container for the semen sample, an incubator, 50 mL plastic tubes, a rack for placing 50 mL plastic tubes at a 45° angle, HTF+ medium (prepared in step 1 or obtained from commercial provider), a centrifuge, and human serum albumin (HSA).
3. Counting of Sperm Cells
NOTE: Sperm cells can either be counted manually50 or using an image cytometer51, as described here. Use an image cytometer, a vortexer, S100 buffer, a SP1-cassette, swim-up purified sperm cells (prepared in step 2).
4. Measurement of Changes in the Free Intracellular Ca2+-concentration ([Ca2+]i) Using Ca2+-fluorimetry (Figure 2)
NOTE: Use a fluorescence plate reader, 384 multi-well plates, Fluo-4 AM, swim-up purified sperm cells (prepared in step 2), compounds of interest as well as positive and negative controls, a automatic repeater pipette, and a 12-channel pipette.
5. Analysis of Data from Ca2+-fluorimetry
6. Measurement of Acrosome Reaction
NOTE: Use an image cytometer, an incubator, fluorescent dyes: PI, FITC-PSA, and Hoechst-33342, compounds of interest as well as positive and negative controls, an immobilizing solution containing 0.6 M NaHCO3 and 0.37% (v/v) formaldehyde in distilled water, an A2 slide, and capacitated swim-up purified sperm cells (prepared in step 2).
7. Analysis of Data from image Cytometry
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 serially diluted concentrations of progesterone, tested in another experiment using the medium-throughput Ca2+-signaling assay. The analysis of the data from the Ca2+-signaling assay is explained in step 5. Representative results from an experiment testing the induction of acrosome reaction in capacitated human sperm cells using a negative control (DMSO) or the two positive controls (progesterone and ionomycin) on the medium-throughput acrosome reaction assay can be seen in the top panel of Figure 3. Quadrant gated scatter plots from the 3 test conditions are seen. The analysis of the acrosome reaction data is explained in step 7.
Table 1: Stock solutions for preparation of HTF+. Stock solutions can be kept and reused for longer periods of time, Glucose and HEPES at -20 °C and the other stock solutions at room temperature. Ensure that all solutions are completely dissolved and well mixed before use.
Table 2: Preparation of HTF+ with 4 or 25 mM HCO3– from stock solutions of salts (prepared in Table 1). Na-Lactate can be added as syrup, 60 % (w/w), or powder. NaHCO3 is added as powder.
Figure 1: Swim-up purification of motile human sperm cells. Left: Tube with HTF+ medium and an aliquot of liquefied semen sample pipetted below the HTF+ medium. Right: After 1 h of swim-up at 37 °C, motile sperm cells have swum up into the HTF+ medium. Immotile and dead sperm cells as well as non-sperm cells remain in the semen below the HTF+ medium. Please click here to view a larger version of this figure.
Figure 2: Diagram of the medium-throughput Ca2+-signaling assay. (a) Sperm cells are loaded with a Ca2+-sensitive fluorophore, washed and aliquots are plated to the wells of a 384-well plate. (b) 384-well plate is positioned in a fluorescence plate reader at 30 °C. (c) Fluorescence is recorded before and after addition of compounds, positive control (progesterone) and negative control (buffer with vehicle). Readouts in ΔF/F0 (%) after addition (arrows) of negative and positive control are illustrated in the bottom of (c). Illustration used with permission from Christian Schiffer. Please click here to view a larger version of this figure.
Figure 3: Assessment of acrosome reaction using image cytometry. (Top panel) Quadrant gated scatter plots from a negative vehicle control (DMSO), and the positive controls progesterone (Prog, 10 µM) and ionomycin (Iono, 2 µM). An increment in cells in the lower right quadrant (acrosome reacted viable cells) is seen when comparing the positive controls to the DMSO control. (Bottom panel) Microscopic images of fluorescently labeled sperm cells, including cells from all four groups. BF = Bright field, Hoechst = Hoechst-33342, PSA = Pisum Sativum Agglutinin, PI = Propidium Iodide. Scale bar = 10 µm. This figure has been modified from Egeberg Palme et al. 20187. Please click here to view a larger version of this figure.
Figure 4: Example of data from Ca2+-fluorimetry. (a) Changes in [Ca2+]i induced by negative control, progesterone and compound A, B, C, and D. (b) Progesterone dose-response curve. Please click here to view a larger version of this figure.
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 individual cells. To obtain such information, experiments with single cell resolution (e.g., as described in50,52) must be employed. Another drawback of the technique is the temporal resolution, which is on the order of seconds. Even though the 50 µL sperm samples are well-mixed when 25 µL of the solutions are added, the measurements can first be initiated after the drawer with the multi-well plate is back inside the fluorescence plate reader. To obtain higher temporal resolution, other techniques must be employed (e.g., a stopped flow fluorimetry13,50). The advantage of the medium-throughput Ca2+-signaling assay, compared to single-cell or single-well methods is that the simultaneous measurement of multiple microwells using a microplate reader allows for fast and easy testing of multiple compounds side-by-side with positive and negative controls (Figure 4)1,2, easy generation of dose-response curves of individual compounds (Figure 4)1,2,3,4,5, assessment of additivity1,2 and synergism3 for two or more compounds, as well as studies of mode of action of compounds though competitive inhibition experiments and the use of specific pharmacological inhibitors (e.g., CatSper inhibitors1,2,3,4,5). Furthermore, by changing the fluorophore, the assay can be employed to examine other intracellular changes (e.g., pHi1,2). Finally, more than one fluorophore may be used at once to simultaneously measure multiple intracellular changes. In the future, the medium-throughput Ca2+-signaling assay could be used to screen compounds of interest for effects on Ca2+-signaling in human sperm (e.g., environmental chemicals or drug candidates) to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive.
The medium-throughput acrosome reaction assay is based on image cytometry, which constitutes an appealing substitute to assess human acrosome reaction compared to manual evaluation using a fluorescence microscope. Staining patterns of intact or reacting/reacted acrosomes are not always clear, and a great intra-individual variation can be the consequence hereof. With image cytometry, images are acquired and analyzed automatically. The masking channel, in this case Hoechst-33342, defines cells and only fluorescent signals from these specific areas are included in the data analysis. Hence, staining of enucleated bodies containing residual cytoplasm is not included in the data analysis. Furthermore, counting statistics is superior to fluorescence microscopy. To count 200 cells through the oculars of the microscope is a challenging and time-consuming task but with image cytometry the analysis of at least 5,000 cells takes less than a few minutes. However, due to the low magnification of the image cytometer, the ability to evaluate the measurements is lost and one will have to bring the stained sample to the fluorescence microscope. Therefore, the inclusion of controls is of outmost importance to validate the measurements. If the controls do not react as expected, the results from an experiment should be discarded. The protocol described here was inspired by work done by Zoppino et al.49, who evaluated human acrosome reaction by flow cytometry. They further described by real time fluorescence microscopy how PSA enters the acrosomal compartment when membrane-fusion pores appear upon induction of acrosome reaction. Once inside the acrosomal compartment, PSA clogs and forms a mark stable for longer periods of time. No fixation or permeabilization is applied in the protocol and therefore the interpretation of the result is clear: PSA inside the acrosomal compartment of a viable sperm cell equals acrosome reaction. As for the Ca2+-signaling assay, the acrosome reaction assay could also be used to assess dose-response curves, inhibition, additivity, and synergism of compounds of interest. In the future, the medium-throughput acrosome reaction assay could also be used to screen compounds of interest for effects on acrosome reaction human sperm (e.g., environmental chemicals or drug candidates) to examine the potential adverse effects on this human sperm cell function or to investigate a possible role as a contraceptive.
The authors have nothing to disclose.
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).
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 |