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Different Approaches to Record Human Sperm Exocytosis

  • Laila Suhaiman
  • Karina Noel Altamirano
  • Alfonsina Morales
  • Silvia Alejandra Belmonte
Protocol
  • 57 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2233)

Abstract

Acrosome reaction is an exocytic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane, releasing the acrosomal content. Given the importance of the acrosome secretion in fertilization, many different methods have been developed to detect the acrosome reaction of sperm. In this chapter, we describe detailed practical procedures to assess the acrosomal status of human spermatozoa. To do this, we resorted to light optical and epifluorescence microscopy, flow cytometry, and transmission electron microscopy. We also itemize the protocol for real-time measurements of the acrosome reaction by confocal microscopy. Further, we discuss the level of complexity, costs, and the reasons why a researcher should choose each technique.

This chapter is designed to provide the user with sufficient background to measure acrosomal exocytosis in human sperm.

Key words

Acrosome exocytosis Human sperm Acrosome reaction Recording sperm secretion 

1 Introduction

Secretion of neurotransmitters, hormones, and enzymes is a fundamental biological activity of the cell and is achieved by vesicular exocytosis, that is, fusion of secretory vesicles with the plasma membrane. Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm exocytosis is essential for egg fertilization. Fertilization is an essential step in sexual reproduction and consists of a carefully orchestrated series of events that culminate with the generation of a genetically unique zygote [1]. Sperm-egg coat penetration, fusion with the egg’s plasma membrane, and finally, fertilization require the release and exposure of cell components resulting from exocytosis of the unique sperm vesicle [2].

1.1 General Features of a Human Spermatozoon

The spermatozoon is a highly polarized cell constituted by a head and a tail or flagellum. The head externally shows two major domains, the acrosomal region and the post-acrosomal region, separated by the equatorial region. It contains a nucleus and an acrosome, which is a Golgi-derived, lysosome-like, very large electron-dense granule covering about 50% of the nucleus in human sperm [3]. The flagellum contains a 9 + 2 array of microtubules, sheath proteins, and mitochondria, which are spirally arranged in the flagellar midpiece, contributing to power its movement. The head and the tail are joined at the neck. The plasma membrane wraps these structures leaving a little cytoplasm inside. Sperm are incapable of synthesizing proteins or nucleic acids. The only purpose of this terminally differentiated cell is to find, fuse, and deliver their genetic information to the egg (Fig. 1).
Fig. 1

Top. Scheme showing general features of the human spermatozoa. The major domains on the sperm head are the acrosomal (a), post-acrosomal region (PA), and the equatorial segment (ES). The flagellum can be divided into three regions: the middle piece, containing the helically wrapped mitochondria, the principal piece, and the end piece. The schematic representation of a sagittal section reveals the detailed structure of the head. The plasma membrane (PM) overlies the outer acrosomal membrane (OAM), in turn the inner acrosomal membrane (IAM), overlies the nuclear envelope. Bottom. Diagram of the sequence of events that occurs in spermatozoa’s head during human sperm acrosome reaction. (a) Represents the head of an intact acrosome sperm as described at the top. (b) After a stimulus, the acrosome swells showing deep invaginations of the OAM. The protruding edges of the invaginations tightly appose to the PM. (c) Represents the stage where fusion occurs between the OAM and PM at multiple sites. As exocytosis proceeds, the acrosomal matrix disperses and hybrid vesicles are sloughed. (d) Finally, when the acrosome is lost, the IAM becomes the limiting membrane of the cell and now the spermatozoa has completed its reaction. The acrosomal plasma membrane fuses with the OAM at the equatorial segment and maintains cytoplasmic integrity in the posterior head. The equatorial segment overlies that region where the inner and outer acrosomal membranes merge. The equatorial region of the sperm is ready to fuse with the oocyte at this point

The large and flat granule covering half of the nucleus, at its apical part, is not just a bag of soluble enzymes, easily released when the outer acrosomal membrane fuses with the plasma membrane. Its content is electron-opaque, given that it contains an important acrosomal matrix required in fertilization. This matrix is a molecular scaffold assembly that is dismantled by a self-regulated mechanism driven, in part, by proteolysis [4].

The acrosome has all the characteristics attributed to secretory vesicles. It undergoes regulated exocytosis when challenged by physiological stimuli, like progesterone or zona pellucida glycoproteins. Although a continuous membrane surrounds the acrosome granule, the membrane consists of different parts that involve distinct functions. The portion overlying the nucleus is termed the inner acrosomal membrane (IAM) and that underlying the plasma membrane is termed the outer acrosomal membrane (OAM). As stated above, the acrosome contains soluble proteins and an insoluble acrosomal matrix, which have their own patterns of release [5, 6]. Soluble factors are readily released once the exocytosis starts, whereas matrix components remain associated for prolonged periods after exocytosis.

1.2 Exocytosis of the Acrosome: Morphological Changes of the Granule

Upon ejaculation in the female genital tract, millions of sperm ascend the uterus. However, only a few pass through the oviduct to reach the ampulla where the fertilization occurs. During this transit, sperm acquire the ability to fertilize eggs through a process defined as capacitation, which consists of a series of physiological and molecular changes that sperm acquire in the female reproductive tract [7]. These changes pertain to the sperm motility pattern, called “hyperactivation,” [8] and to their ability to undergo sperm granule exocytosis. In the proximity of the egg, progesterone and zona pellucida glycoproteins stimulate mammalian sperm to release the contents of the acrosome granule, a key event in fertilization. Acrosomal exocytosis is an all-or-nothing event that involves the opening of multiple fusion pores between the outer acrosomal membrane and the plasma membrane. This Ca2+-regulated exocytosis is known as “sperm acrosome reaction.” Initially, the importance of exocytosis in mammalian fertilization was unclear, but the phenomenon soon became recognized as a prerequisite for fertilization.

Acrosome reaction involves the prominent rearrangement of membranes in the sperm head. In resting sperm, the majority of cells show an acrosome with electron-dense content and a flat OAM close and parallel to the plasma membrane. Even though, these two membranes do not interact until the sperm is stimulated in the proximity of the mature oocyte to undergo the acrosome reaction. After stimulation with a calcium ionophore or progesterone [9, 10], 30–40% of the sperm show morphologically altered acrosomes. Strikingly, the morphology of the equatorial region of the acrosome is similar to that observed in non-stimulated cells, meaning that this region remains intact. Some acrosomes undergo a simple swelling; however, in many cells, the OAM shows a wavy surface. The protrusions of this membrane occasionally come in contact with the plasma membrane and result in invaginations that are very deep (Figs. 1 and 5). Some cells appear to have vesicles inside the acrosome. Finally, a small group of cells show vesiculated or lost acrosomes, this state is known as reacted. Therefore, in response to exocytosis inducers, a granule first swells to get into contact with the cell plasma membrane, second, it gets attached to the plasma membrane and fuses with it. Finally, it detaches entirely, together with the portion of the plasma membrane that surrounds it. These events are coupled to a complex calcium signaling [11, 12].
Fig. 2

The micrograph shows that Coomassie Blue G-250-stained acrosome-intact sperm are easily discernible from acrosome-reacted sperm. Human sperm were washed, fixed, and stained with the dye. Arrows indicate acrosome-intact sperm. Asterisk indicates acrosome-reacted sperm that shows negligible staining in the acrosomal region as compared to the acrosome-intact sperm

In most secretory processes, fusion pore opening and expansion lead to the release of granule contents and the incorporation of the acrosomal membrane into the plasma membrane. In contrast, in mammalian sperm, pore opening and expansion cause the vesiculation of the acrosome. Acrosomal swelling and OAM deformation are important to delineate the membrane domains where pore expansion will lead to the release of hybrid vesicles (Figs. 1 and 5). Reproductive biologists have not yet reached a consensus about crucial issues including this remarkable deformation of the acrosomal granule occurring during the acrosome reaction, but it is thought to be related to lipid remodeling.

1.3 Kinetics of the Acrosome Granule Exocytosis

Most of the knowledge of exocytosis comes from scientific reports on secretion in neurons and neuroendocrine cells. These cells contain several small vesicles that fuse quickly with the plasma membrane. Much less is known about secretion of the large granules stored in exocrine cells, such as pneumocytes and acinar and salivary cells. There is an important correlation between granule size and secretion kinetics [13]. The acrosome reaction is not an exception to this rule: large acrosomal granules exhibit slow (minutes) kinetics of release [10]. Results from several laboratories have shown that the percentage of reacted sperm increases with the time of incubation in the presence of physiological and pharmacological stimuli [14, 15, 16]. The kinetics of the process is measured in minutes and depending on the experimental conditions, it may require about 1 h to reach a plateau. Sosa et al. [10] measured the kinetics of human sperm exocytosis using different approaches. The swelling of the acrosomal granule, which precedes exocytosis, was a slow process (t½ = 13 min). When the swelling was completed, the fusion pore opening occurred rapidly (t½ = 0.2 min). Therefore, the acrosomal swelling is the slowest step and it determines the kinetics of acrosome reaction. After swelling is completed, the efflux of calcium from intracellular stores triggers fusion pore opening and the release of hybrid vesicles in seconds.

1.4 Why Is This Chapter in This Book?

Exocytosis of the acrosomal vesicle is unique, owing to the size and morphology of the granule, as well as the nature of the acrosomal contents; consequently, its exocytosis is a slow event.

Despite the singular morphological and functional features, all of the fusion-related molecules involved in the acrosome reaction were initially found in somatic cells and implicated in various membrane fusion events. Therefore, sperm share their basic fusion machinery and regulatory components with all other eukaryotic cells, but their differences have to be highlighted [17, 18, 19, 20, 21, 22].

Different cell types carry different secretory granules, such as neuronal, neuroendocrine, endocrine, and exocrine cells. Regulatory exocytosis varies among these cells in terms of cargo, kinetics, probability, and modality of release [12]. To release their contents, secretory vesicles must travel from a cytosolic compartment toward the plasma membrane. Because of its size and shape, the acrosome cannot travel to contact the plasma membrane. The acrosome reaction shares many features with exocytosis in other cell types, however, it differs at the following points:
  1. 1.

    Sperm contains only one secretory vesicle that overlies the anterior half of the nucleus in the apical region of the head instead of many secretory granules;

     
  2. 2.

    This granule has a unique secretion mode that differs from full collapse, kiss, and run, or compound exocytosis. On the contrary, the sperm acrosome reaction involves fusion at multiple points of the OAM and the overlying plasma membrane. Thus, many fusion pores are formed instead of one as in secretory somatic cells;

     
  3. 3.

    In contrast to full-collapse exocytosis where the release of granular contents into the extracellular medium causes the incorporation of their membranes into the plasma membrane, acrosome reaction is characterized by the release of hybrid vesicles (half OAM and half plasma membrane) into the media with membrane loss.

     
  4. 4.

    Acrosome reaction is a tightly regulated and irreversible process with unknown membrane recycling. Once the granule releases the acrosomal contents, the cell modifies the membrane components entirely, exposing the IAM to the extracellular medium and keeping the equatorial segment intact (Fig. 1).

    The end point of most studies involving acrosome reaction is the staining of cells at different time points after stimulation to calculate the percentage of reacted sperm. This experimental setting measures three time components: (1) the time required for a stimulated sperm to activate the molecular machinery of membrane fusion, (2) the time required for the actual opening of fusion pores, and (3) the time required for the expansion of fusion pores to release the acrosomal contents. It also includes the probability of each cell to respond to the stimulus received. It is essential to clarify that not all human sperm react at the same time, but in fact a small percentage (10–30%) undergo the acrosome reaction [10, 16, 23]. The reason for this asynchrony is not understood.

    Spermatozoa are terminal cells lacking almost all organelles, transcriptional and translational activities. From the point of view of intracellular trafficking, sperm are specialized for a single membrane fusion event, the exocytosis of the acrosome granule. This feature is particularly useful to study exocytosis in isolation. At the same time, their inability to synthesize proteins constitutes the biggest obstacle when trying to apply classic cell biology and biochemistry approaches to sperm. Our laboratory has successfully contributed to establishing two techniques to overcome this limitation: controlled plasma membrane permeabilization with pore-forming toxins, like streptolysin or perfringolysin O, and delivery of permeable proteins [17, 24, 25, 26, 27, 28, 29, 30]. The latter allows sophisticated molecular studies of the pathways elicited by well-established AR inducers because it grants access to intracellular compartments in non-permeabilized cells.

    Given the particular characteristics of sperm exocytosis discussed above, methods commonly used to measure secretion in other cells cannot be used for this purpose. Here, we provide detailed protocols for studying human sperm-regulated exocytosis at a defined time or at real time by using different approaches.

     

2 Materials

Ultrapure water must be used to dilute all reagents. Prepare it by purifying deionized water to attain a sensitivity of 18 M Ω cm at 25 °C. Use analytical-grade reagents. Follow all waste disposal regulations when discarding materials.

2.1 Human Tubal Fluid (HTF Media)

For 1 L of HTF weigh 5.94 g/L NaCl, 0.35 g/L KCl, 0.05 g/L MgSO4·7H2O, 0.05 g/L KH2PO4, 0.3 g/L CaCl2·2H2O, 2.1 g/L NaHCO3, 0.51 g/L d-glucose, 0.036 g/L Na pyruvate, 2.39 g/L Na lactate, 0.06 g/L penicillin, 0.05 g/L streptomycin, and 0.01 g/L phenol red (see Notes 13).

2.2 Hepes Buffer- Ethylene Glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic Acid (HB-EGTA)

250 mM sucrose, 20 mM HEPES free acid, 0.5 mM EGTA, pH 7. Dissolve all the reagents in water at room temperature by stirring (see Note 4). Adjust pH with 1 N KOH.

2.3 Phosphate-Buffered Saline

For 1 L of PBS (1×) weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4. Adjust pH to 7.4 with HCl. Finally, add distilled water to a total volume of 1 L.

2.4 Exocytosis Inducers Used for Non-permeabilized Spermatozoa (See Note 5)

  1. 1.

    A23187 (see Note 6). For 100 mM stock solution 1: 1 μL of 100 mM stock solution 1–9 μL of dimethyl sulfoxide (DMSO) to yield a 10 mM solution (stock solution 2). Tip off 2.5 μL of the stock solution 2 and pour it on 47.5 μL of HTF without bovine serum albumin (BSA) to yield a final concentration of 0.5 mM (Stock solution 3). Add 1 μL of the stock solution 3 to the experimental condition tube containing 49 μL of sperm suspension to obtain a final concentration of 10 μM. Stock solutions can be stored up to 3 months at −20 °C (see Note 7).

     
  2. 2.

    Progesterone (Pg, see Note 8). Prepare 10 mM stock solution in absolute ethanol and gently swirl to dissolve. Tip off 2.5 μL of the stock solution and pour it on 47.5 μL of HTF without albumin to yield a final concentration of 0.5 mM. Add 1.5 μL of 0.5 mM Pg solution in 48.5 μL of sperm suspension in HTF with BSA to obtain a final concentration of 15 μM. Store aliquots at −20 °C. Avoid repeated freeze–thaw.

     
  3. 3.

    Diacylglycerol (DAG, see Note 9). Prepare a 0.5 mM solution in HTF without BSA (dissolve 1 μL of a 25 mM DMSO stock solution in 49 μL of HTF without BSA). To obtain a final concentration of 10 μM DAG in the experimental tube, add 1 μL of the 0.5 mM solution in HTF to 49 μL of sperm suspension.

     
  4. 4.

    Phorbol 12-Myristate 13-Acetate (PMA, see Note 10). Prepare a 1 M stock in DMSO, apply a N2 stream, and store at −20 °C. Add 0.5–49.5 μL of DMSO to obtain a 0.01 M solution. From this stock, prepare additional dilutions in DMSO until a 10 μM PMA solution is obtained. Add 1 μL from the 10 μM stock to 49 μL of sperm suspension to get a final concentration of 200 nM.

     
  5. 5.

    Sphingosine-1-Phosphate (S1P). Prepare a solution by adding 995 μL of methanol and 5 μL of water and add it to the vial containing sphingosine-1-phosphate (S1P) (1 mg), provided by the manufacturer. Heat at 50 °C in a water bath, vortex, and then sonicate in a water bath sonicator to dissolve the contents. Label ten glass vials covered with aluminum foils or use amber glass vials. Aliquot 100 μL into the glass vials. Dry to powder under a stream of N2 gas. Seal vials. Freeze and store at −80 ºC. Resuspend dried aliquots in 100 μL HTF supplemented with 0.5% fatty acid free BSA to yield 2.6 mM S1P stock solution [31].

     
  6. 6.

    Soluble Human Recombinant Zona Pellucida (ZP, see Note 11). We have performed functional experiments to assess ZP's ability to induce acrosomal exocytosis for isoforms 2, 3, and 4 (unpublished data). To perform functional assays, we incubate 1 × 106sperm with 1 μg/μL final concentration of ZP2 (negative control), ZP3, or ZP4.

     

2.5 Exocytosis Inducers Used for Permeabilized Spermatozoa (See Note 12)

  1. 1.

    Calcium. Dissolve 2.77 mg CaCl2 in 1 mL ultrapure water to obtain a 25 mM stock solution. Tip off 1 μL and pour it on 49 μL of sperm cells suspension in HB-EGTA to yield a final concentration of 0.5 mM (see Note 13).

     
  2. 2.

    Diacylglycerol. Prepare this solution as described under Subheading 2.4, item 3 (for non-permeabilized cells), an exception is the final stock solution which must be prepared in HB-EGTA.

     
  3. 3.

    Phorbol 12-Myristate 13-Acetate. Prepare this solution as described under Subheading 2.4, item 4. The final stock solution must be prepared in HB-EGTA.

    For further information see Note 14.

     

2.6 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin

Prepare a 5 mg/mL PSA stock solution in PBS and store aliquots at −20 °C. PSA working solution: Dilute 1 μL of PSA stock solution in 199 μL of PBS (intact sperm) or HB-EGTA (permeabilized sperm). This dilution must be used directly to stain the samples. Wrap the tubes with aluminum foil to protect from light.

2.7 Coomassie Blue G250 Solution

Weigh 0.11 g Coomassie Blue G 250 and dissolve in methanol/glacial acetic acid/distilled water (v/v 25:5:20 mL).

2.8 Four Percent Paraformaldehyde Solution

Weigh 4 g PAF and 0.5 g NaOH. Add 90 mL deionized water. Transfer to an Erlenmeyer flask and cover it with aluminum paper. Heat the solution on a magnetic stirrer under fume hood until the solution is transparent (60 °C). Adjust pH to 7. Add 10 mL 1× PBS to reach 100 mL as final volume. Freeze aliquots.

2.9 Ammonium Acetate (0.1 M)

Weigh 0.07 g ammonium acetate (CH3CO2NH4) and add 10 mL of deionized water.

2.10 Sodium Cacodylate Solution

Weigh 21.40 g sodium cacodylate ((CH3)2AsO2Na·3H2O) and add 1 L of deionized water to reach 0.1 M final concentration, pH 7.4.

2.11 Glutaraldehyde Solution (2.5%)

Use electron microscopy grade 25% glutaraldehyde in sealed ampoules. Add 1 mL of 25% glutaraldehyde to a solution of 9 mL sodium cacodylate (0.1 M, pH 7.4).

2.12 Osmium Tetroxide Aqueous Stock Solution

Work under a fume hood and use nitrile gloves. Open a sealed glass ampoule of 0.5 g OsO4 and add it to deionized water. Complete with water to reach a final volume of 25 mL (2% final concentration).

2.13 Uranyl Acetate Aqueous Solution

Weigh 1 g uranyl acetate and add to 50 mL of ultrapure water by stirring to reach a final concentration of 2%.

2.14 Lead Citrate Solution

Add 0.1 g NaOH and 0.18 g lead citrate to 25 mL of distilled and boiled water.

3 Methods

3.1 Sample Collection

  1. 1.

    Human semen samples should be obtained by masturbation and ejaculated into a clean, wide-mouthed container made out of glass or plastic, from a batch that has been confirmed to be non-toxic for spermatozoa (see Note 15).

     
  2. 2.

    Label the specimen container with an identification number, the date, and time of collection.

     
  3. 3.

    Place the specimen container in an incubator (37 °C, 5% CO2) to liquefy the semen (30 min to 1 h) (see Note 16).

     
  4. 4.

    After liquefaction, evaluation of semen quality must be done (see Note 17).

     

3.2 Swim-Up (See Note 18)

  1. 1.

    Mix the semen sample well (see Note 19).

     
  2. 2.

    Place 1 mL of semen in a 5-mL polypropylene tube (12 × 75 mm). Alternatively, a conical centrifuge tube can be used.

     
  3. 3.

    Gently layer 1.2 mL of BSA-supplemented medium over the semen sample. Alternatively, pipette the semen carefully into the BSA-supplemented culture medium.

     
  4. 4.

    Place the tubes at 45° angle, to increase the surface area of the semen-culture medium interface, and incubate for 1 h at 37 °C.

     
  5. 5.

    Return the tube to the upright position gently and remove the uppermost 1 mL of medium. This will contain highly motile sperm cells.

     
  6. 6.

    Mix the recovered cells well for assessment of sperm concentration (see Notes 20 and 21).

     
  7. 7.

    The specimen may be used directly or after capacitation for research purposes.

     

3.3 Capacitation of Spermatozoa

For acrosome reaction assays, spermatozoa must be capacitated (see Note 22).
  1. 1.

    Prepare the HTF-BSA capacitation-inducing medium fresh for each assay (see Note 3).

     
  2. 2.

    Warm up the medium to 37 °C before use, preferably in a 5% (v/v) CO2-in-air incubator.

     
  3. 3.

    Dilute the motile sperm population obtained from swim-up to 10 × 106 cells/mL using fresh warm HTF-BSA medium.

     
  4. 4.

    Incubate the sperm suspensions for at least 3 h at 37 °C in an atmosphere of 5% (v/v) CO2-in-air incubator to induce capacitation (see Note 23) [32].

     

3.4 Functional Assays

Once capacitation time has ended, cells can be treated with inhibitors or stimulants of acrosomal exocytosis immediately, or permeabilized. In both cases, cells can be treated as follows:
  1. 1.

    Prepare experimental tubes, each containing the volume established for each reagent from stock solutions (stimulants or inhibitors of different components of the signal transduction cascade, see Subheading 2.4). Identify different conditions with numbers .

     
  2. 2.

    Add to each tube the volume of sperm suspension required to reach 50 μL (7–10 × 106/mL motile spermatozoa, 350,000 to 500,000 cell/condition).

     
  3. 3.

    Mix gently with a pipette changing the tip for each condition.

     
  4. 4.

    Incubate all the tubes for 15 min at 37 °C, 5% CO2/95% air.

     
  5. 5.

    If the cells are treated with an inhibitor, subsequently add the microliters stated in Subheading 2.4 for the stimulus to be used and incubate for an additional 15 min at 37 °C, 5% CO2/95% air.

     
  6. 6.

    For control conditions, incubate the sperm without the addition of any reagent (see Note 24).

     
  7. 7.

    After that, 10 μL of each reaction mixture must be spotted on 8- or 12-well slides (microscope slides, coated with Teflon, size: 75 × 25 × 1 mm).

     
  8. 8.

    Air-dry the spots.

     
  9. 9.

    When dried, fix/permeabilize the cells covering the slide with 1 mL of ice-cold methanol for 1 min.

     
  10. 10.

    Wash four times × 5 min each with distilled water by placing 1.5 mL on the slide after turning over the slide to eliminate the water of the previous wash (see Note 25).

     
  11. 11.

    Using a conventional transillumination microscope check if the preparation is clean enough.

     

3.5 Permeabilization of Spermatozoa (See Note 26)

  1. 1.

    Recombinant streptolysin O (SLO) was obtained from Dr. Bhakdi (University of Mainz, Mainz, Germany).

     
  2. 2.

    Wash the sperm suspension 2× with ice-cold PBS (use the spermatozoa recovered from swim-up; capacitate and dilute to 7–10 × 106, as described in Subheadings 3.2 and 3.3.

     
  3. 3.

    Suspend the pellet by gently pipetting in a volume of cold PBS equal to the original one.

     
  4. 4.

    Before using a new batch of the protein performe a dose-response curve to determine the optimal SLO concentration to be used.

     
  5. 5.

    Add 0.7 μL of pre-activated SLO (300 U/mL) per 100 μL of sperm suspension to get a final concentration of 2.1 U/mL (see Note 27).

     
  6. 6.

    Incubate for 15 min at 4 °C.

     
  7. 7.

    Remove the remaining non-bound toxin from the cell suspension by washing once with cold PBS.

     
  8. 8.

    Resuspend the pellet in ice-cold HB-EGTA (see Subheading 2.2) containing 2 mM dithiothreitol (DTT) to provide a reducing environment.

     
  9. 9.

    Label the tubes with numbers and add to each tube the volume of stimulants or inhibitors as described in Subheading 2.5.

     
  10. 10.

    Aliquot sperm suspension (prepared in HB-EGTA plus 2 mM DTT) in the experimental tubes containing reagents to a final volume of 50 μL. First add an inhibitor and incubate with the cells for 15 min at 37 °C, 5% CO2/95% air. After that, the stimulus must be added and tubes incubated for an additional 15 min under the same conditions.

     
  11. 11.

    Include in all experiments negative (no stimulation) and positive controls (stimulated with 0.5 mM CaCl2 rendering 10 μM free calcium (see Subheading 2.5, item 1)).

     
  12. 12.

    Finally, 10 μL of each reaction mixture must be spotted on 8- or 12-well slides (microscope slides, coated with Teflon, size: 75 × 25 × 1 mm).

     
  13. 13.

    Let the spots air-dry.

     
  14. 14.

    When dried, fix/permeabilize the cells covering the slide with 1 mL of ice-cold methanol for 1 min.

     
  15. 15.

    After that, follow the protocol for assessing the acrosomal exocytosis as described in Subheading 3.4, steps 711.

     

3.5.1 SLO Pre-activation

  1. 1.

    Add 1 volume of 6 mM DTT to 2 volumes of 450 U/mL SLO, obtaining a final SLO concentration of 300 U/mL.

     
  2. 2.

    Incubate the mixture at 37 °C for 30 min. After that incubation period, store the protein on ice until use.

     

3.5.2 SLO Dose–Response Curve (See Note 28)

  1. 1.

    Before using a new batch of the protein, a dose–response curve must be generated. Generate a curve from 0.1 U/mL to 10 U/mL.

     
  2. 2.

    Evaluate the percentage of permeabilized cells by eosin yellowish staining (see Note 29).

     
  3. 3.

    Choose the SLO dilution that renders 70–80% of permeabilized cells (meaning the percentage of cells where eosin has entered via membrane pores).

     
  4. 4.

    Perform a functional assay as described in Subheading 3.4 using calcium as a stimulus.

     
  5. 5.

    Aliquots of concentrated stock of SLO (e.g., 25,000 U/mL) must be stored at −80 °C. Avoid repeated freeze–thaw (see Note 30).

     

3.6 Assessment of the Acrosome Reaction (See Note 31)

3.6.1 Visualization of Acrosomal Status Using Light Microscopy. Coomassie Blue Staining Method (See Note 32)

Follow this protocol for the cells obtained and treated as described in step 6 of Subheading 3.4.
  1. 1.

    Centrifuge the sperm suspension at 700 × g for 3 min.

     
  2. 2.

    Wash the pellet with HTF (RT) without BSA and resuspend it in the same media (10 × 106 cells/mL).

     
  3. 3.

    Prepare experimental tubes by adding the volume of sperm suspension required to reach 1 × 106 motile spermatozoa/mL per condition (100 μL).

     
  4. 4.

    Add the volume established for each inducer from stock solutions (stimulants, see Subheading 2.4). Identify different conditions with numbers.

     
  5. 5.

    Mix gently with a pipette, changing the tip for each condition.

     
  6. 6.

    Incubate all the tubes for 15 min at 37 °C, 5% CO2/95% air.

     
  7. 7.

    Centrifuge sperm suspension at 2600 × g for 3 min. Wash the pellet with HTF without BSA.

     
  8. 8.

    Suspend the pellet in 4% PAF (see Subheading 2.8) (100 μL). Incubate the sperm for 1 h at 4 °C.

     
  9. 9.

    Centrifuge at 1700 × g for 2 min.

     
  10. 10.

    Wash the cells once first with PBS 1× and then twice with 0.1 M ammonium acetate pH 9.

     
  11. 11.

    Centrifuge the cells at 700 × g for 3 min.

     
  12. 12.

    Prepare sperm smears with 100 μL of suspension on poly-l-lysine-coated microscope slides (see Note 33).

     
  13. 13.

    Allow the slides to air-dry.

     
  14. 14.

    Inspect the smears under a phase-contrast microscope (×400).

     
  15. 15.

    Ensure that the spermatozoa are evenly distributed on the slides without clumping.

     
  16. 16.

    Wash the slides with distilled water for 5 min.

     
  17. 17.

    Incubate with methanol for 5 min.

     
  18. 18.

    Wash with distilled water for 5 min.

     
  19. 19.

    Let the slides air-dry and then cover them with a Coomassie blue solution (see Subheading 2.7) for 10 min.

     
  20. 20.

    Wash the excess of Coomassie blue by immersing the slides in distilled water at RT. For this purpose, use a glass coplin jar.

     
  21. 21.

    Let the slides air-dry.

     
  22. 22.

    Drop Mowiol (prepared as the manufacturer instructions) on the slide, place over a coverslip, and observe under light optical microscope.

     
  23. 23.

    Scoring (see Note 34 and Fig. 2.)

     

3.6.2 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin Assessment of Acrosomal Status by Indirect Staining Method Using Epifluorescence Microscopy (See Notes 35 and 36)

Follow this protocol for the cells obtained and treated as described in Subheadings 3.4 or 3.5.
  1. 1.

    Prepare a 50 μg/mL solution of FITC-PSA in 1× PBS (see Subheading 2.6see Note 35.

     
  2. 2.

    Pour 10 μL of the lectin dilution solution on each spot.

     
  3. 3.

    Incubate in a moist chamber in the dark for 40 min at RT.

     
  4. 4.

    Wash the excess of lectin by immersing the slide in distilled water for 20 min at 4 °C. For this purpose, use a glass coplin jar with lid covered with aluminum foil to keep in dark.

     
  5. 5.

    Allow drying in a dark chamber.

     
  6. 6.

    View the slide using a microscope equipped with epifluorescence optics at 600× magnification with oil immersion at 450–490 nm excitation.

     
  7. 7.

    Categorize the spermatozoa as described in Note 36.

     
  8. 8.

    Tally the number in each acrosomal category (AI and AR) with the aid of a laboratory counter.

     
  9. 9.

    Score at least 300 cells in order to achieve an acceptably low sampling error. Include in all experiments negative (no stimulation) and positive controls (stimulated with 10 μM A23187 or 15 μM Pg). For permeabilized sperm, use calcium as positive control as indicated in Subheading 2.5, item 1 (see Note 37).

     
  10. 10.

    For each experiment, calculate acrosomal exocytosis indexes as described in Note 38.

     

3.6.3 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin Assessment of Acrosomal Status by Indirect Staining Method Using Flow Cytometry

  1. 1.

    Cells can be stained by using the same technique in suspension as described in Subheading 3.6.2 and scored by flow cytometry.

     
  2. 2.

    Wash the sperm suspension once with warm HTF (no BSA addition) for intact sperm. In case of permeabilized cells, resuspend them in HB-EGTA. Use 100 μL/per condition of spermatozoa recovered from swim-up, capacitated and diluted to 7–10 × 106 as described in Subheadings 3.2 and 3.3. We recommend creating multiple aliquots of different cell conditions in order to obtain measurements in duplicate.

     
  3. 3.

    Follow this protocol from cells obtained and treated as described in Subheadings 3.4 or 3.5.

     
  4. 4.

    Centrifuge at 1700 × g for 3 min.

     
  5. 5.

    Fix/permeabilize with 100 μL of cold methanol for 1 min.

     
  6. 6.

    Centrifuge at 1700 × g for 3 min.

     
  7. 7.

    Wash the cells 1–3 times by resuspending them in 300 μL of distilled water per tube and mix them gently. Centrifuge the samples at 700 × g for 3 min and aspirate the supernatant.

     
  8. 8.

    Prepare a 50 μg/mL solution of FITC-coupled PSA in 1× PBS (see Subheading 2.6).

     
  9. 9.

    Add 50 μL of the lectin dilution solution to each tube. Resuspend by pipetting gently.

     
  10. 10.

    Incubate in the dark for 40 min at RT.

     
  11. 11.

    Centrifuge at 700 × g for 3 min and discard the supernatant.

     
  12. 12.

    Wash the excess lectin 2× by adding 500 μL of distilled water and then centrifuge at 700 × g for 3 min. Discard the supernatant. Protect the tubes from light.

     
  13. 13.

    Suspend in 200 μL PBS 1× and run each sample condition in flow cytometer.

     
  14. 14.

    As an auto fluorescence control, one aliquot of sperm must be left with no lectin addition.

     

3.6.4 Fluorescein-Isothiocyanate-Coupled Pisum Sativum Agglutinin Assessment of Acrosomal Status by Direct Staining Method Using Epifluorescence Microscopy and Flow Cytometry (See Note 39)

Once capacitation time has ended, cells can be treated with inhibitors or stimulants of acrosomal exocytosis immediately.
  1. 1.

    Wash the sperm suspension 1× with warm HTF (no BSA addition) for intact sperm. In case of permeabilized cells, resuspend them in HB-EGTA. Add 5 μg/mL of FITC-PSA. Use 100 μL/per condition of spermatozoa recovered from swim-up, capacitated and diluted to 7–10 × 106, as described in Subheadings 3.2 and 3.3.

     
  2. 2.

    As an auto fluorescence control, one aliquot of sperm should be left with no FITC-PSA addition.

     
  3. 3.

    Other aliquots: incubate with inhibitors for 15 min at 37 °C. Protect them from light by covering the tubes with aluminum foil.

     
  4. 4.

    Incubate with stimulants for additional 15 min at 37 °C.

     
  5. 5.

    Wash twice each condition with either HTF or HB-EGTA.

     
  6. 6.

    Spin down the cells at 700 × g for 3 min.

     
  7. 7.

    Resuspend pellets with 2% PAF.

     
  8. 8.

    Incubate for 15 min at 4 °C.

     
  9. 9.

    Spin down for 3 min at 3300 × g.

     
  10. 10.

    Resuspend pellets in 200 μL 1× PBS (see Note 40).

     
  11. 11.

    Analyze conditions (10.000 events per condition) in a flow cytometer (we analyze the data by FlowJo software). Keep the cells at 4 °C protected from light until they are scored in the cytometer (see Note 41).

     
  12. 12.

    It is very important to vortex each sample before running in the cytometer.

     
  13. 13.

    The gating scheme must be set up in the instrument prior to data acquisition. First, run the control condition (no PSA addition) to gate the cell population and discard cellular debris.

     
  14. 14.

    Then the rest of the conditions can be run according to the experiment.

     
  15. 15.

    Dot plot graphs of a representative experiment are shown in Fig. 4.

    To assess exocytosis by epifluorescence microscopy, cells obtained at step 10 of this protocol can be spotted on 8- or 12-well slides. Check them under the epifluorescence microscope to evaluate the staining pattern. If the fluorescence is faint, stain again as indicated in Subheading 3.6.2. In the direct method, fluorescent acrosomes are considered reacted and no fluorescent acrosomes are scored as intact.

     
Fig. 3

Micrograph of human spermatozoa showing indirect FITC-PSA staining patterns. The lectin stains the intact acrosome (arrows). The micrograph also shows spermatozoa that have lost the acrosome (reacted, asterisk)

3.6.5 Real-Time Measurements of the Acrosome Reaction (See Note 42)

  1. 1.

    Immobilize the capacitated sperm (5 × 106 cells/mL) on poly-l-lysine (0.01% w/v) precoated cover slides (see Note 33).

     
  2. 2.

    Mount the samples in a chamber at 37 °C and overlay them with HTF-BSA medium supplemented with 5 mg/mL FITC-PSA (see Note 43).

     
  3. 3.

    Perform image analysis by using the software Image J (National Institutes of Health, http://rsb.info.nih.gov/ij/) see Note 44.

     

3.6.6 Assessment of Acrosome Reaction and Ultrastructure of Sperm Using Transmission Electron Microscopy (See Note 45)

This protocol can be performed from the cells obtained and treated as described in Subheadings 3.4 or 3.5 as stated in Zanetti and Mayorga [9].
  1. 1.

    After washing with 1× PBS, centrifuge the samples at 1700 × g for 2 min to obtain at least a 20–25 μL of pellet.

     
  2. 2.

    For fixation, suspend the sperm pellet in 200 μL of 2.5% glutaraldehyde in sodium cacodylate (see Subheading 2.11). Incubate for 1 h to ON at 10 °C.

     
  3. 3.

    Wash fixed sperm twice by suspending in sodium cacodylate for 15 min at 10 °C (see Subheading 2.10).

     
  4. 4.

    Fix the samples with 1% osmium tetroxide in sodium cacodylate for 2 h, at RT under a fume hood (see Subheading 2.12).

     
  5. 5.

    Wash the samples three times by suspending in sodium cacodylate for 15 min at 10 °C and spin down in a microfuge.

     
  6. 6.

    Dehydrate the pellets with increasing concentrations of acetone (50%, 70%, 95%, 100% and 100%), for 15 min in each step.

     
  7. 7.

    Embed the pellet with an epoxy resin (preferentially with low viscosity) gradually, with a change in resin/acetone (1:1 v/v) for 2 h. Then add pure resin and let embed ON at RT.

     
  8. 8.

    Polymerize in an oven at 70 °C for 48 h to obtain a hard block.

     
  9. 9.

    Trim the block and section with an ultramicrotome. Collect the grey interference color sections (60-nm thickness) on 300 mesh copper grids.

     
  10. 10.

    Contrast the sections mounted on the grids with a solution of uranyl acetate (see Subheadings 2.13 and 2.14) for 5 min and then with a solution of lead citrate for 1 min.

     
  11. 11.

    Now, grids are ready to be observed at TEM.

     
  12. 12.

    Count at least 100 cells per condition. Acrosomal patterns should be scored and classified as intact, reacted (vesiculated and lost), and swollen (enlarged and deformed) (Fig. 5).

     

4 Notes

  1. 1.

    Use a glass beaker and add the reagents one at a time in water at RT while the magnetic stir bar is working. Make sure that each reagent is completely dissolved before adding the next one. HTF must be sterilized using aseptic processing techniques (filtration). Once prepared, the aliquots can be stored (15 mL) at 2–8 °C until further use. Do not freeze the complete HTF or expose to temperatures greater than 39 °C. To freeze and use aliquots later, combine all but NaHCO3 and freeze at −70 °C. For experiment, thaw an aliquot and add appropriate NaHCO3. Let sit in 5% CO2 at 37 °C ON.

     
  2. 2.

    The medium uses NaHCO3 as a buffering system. This is specifically designed for use in a CO2 incubator. The bottle/tube of HTF medium should be loosely capped to allow for the exchange of gas and pH equilibration. Incubate HTF in 5% CO2 overnight at 37 °C. Check to ensure the pH is between 7.4 to 7.6.

     
  3. 3.

    Human Tubal Fluid media is used to perform the swim-up technique and to isolate motile fraction of a semen sample. To perform acrosome reaction assays, the sample needs to be capacitated, which is achieved in vitro by incubation of the motile fraction recovered with HTF supplemented with 5 mg/mL bovine serum albumin (BSA) for at least 3 h [32].

     
  4. 4.

    A 1 M HEPES-free acid solution (954 mg in 4 mL of water) is clear and colorless, with pH approximately between 5.0 and 6.5 at 20 °C. Adjust pH with 1 N KOH.

     
  5. 5.

    The reagents we currently use to induce exocytosis in intact cells are able to penetrate the cell by different mechanisms; they are either permeant, able to open pores in the plasma membrane, or affect the function of some membrane surface proteins, for example, sphingosine 1-phosphate (S1P) receptors. Here, we provide a list of some reagents we usually use to analyze signal transduction cascades involved in exocytosis in human spermatozoa.

     
  6. 6.

    It is a calcium ionophore commonly used to induce exocytosis of the sperm granule.

     
  7. 7.

    DMSO concentration cannot be greater than 0.5% per experimental condition (it may produce cellular damage at higher concentrations).

     
  8. 8.

    Pg is the most widely used physiological inducer of the acrosome exocytosis.

     
  9. 9.

    DAG is an allosteric activator of protein kinase C (PKC) and induces the acrosomal exocytosis in intact and in permeabilized sperm [25, 31]. Storage: diacylglycerols are conveniently stored in chloroform solutions in glass vials with Teflon-lined caps at −20 °C. Avoid plastic when handling chloroform solutions. Delivery to cells: dry DAG aliquots in chloroform, using a stream of N2. Dissolve the residue in an appropriate volume of DMSO, then dilute to the desired concentrations using aqueous medium.

     
  10. 10.

    PMA induces the acrosomal exocytosis. It has a structure analogous to DAG and can also activate PKC [27, 31]. PMA is soluble in DMSO. Prepare a stock solution and store in the freezer, protected from light.

     
  11. 11.

    Zona Pellucida. The zona pellucida are glycoproteins known as physiological inducers of the acrosomal exocytosis. In humans, there are four isoforms that are secreted by oocyte in order to build a specialized form of an extracellular matrix [33]. Human recombinant ZP proteins were obtained from Dr. Mayel Chirinos (Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico DF, Mexico). The protocol to obtain these proteins can be found in their publication [34]. The concentration of the single proteins to be used in non-permeabilized cells depends on each production batch.

     
  12. 12.

    We routinely use some recombinant permeant proteins involved in the signaling pathway leading to acrosome reaction, produced at our laboratory, as inducers or inhibitors of exocytosis. We cannot include the protocols to produce the proteins in this chapter, given that they exceed the topics to be developed here. If the readers are interested in that protocols, they can resort to the following bibliography [26, 30, 35].

     
  13. 13.

    This dilution renders 10 μM free calcium, estimated by MAXCHELATOR, a series of programs for determining the free metal concentration in the presence of chelators available at http://maxchelator.stanford.edu.

     
  14. 14.

    The reagents used in permeabilized cells are different from those described in Subheading 2.4 Given that ion gradient is lost due to plasma membrane pores, these reagents become inactive. These reagents cannot be used as they are known to affect plasma membrane channels (because the plasma membrane is perforated). Further these inducers cannot be used as they interact with plasma membrane receptors. If an enzyme inhibitor is chosen for a defined signal transduction pathway, the component does not need to be permeant. Prepare the reagents following the manufacturer instructions and always generate a dose-response curve for the cells before use. Spermatozoa usually require inhibitors or stimulant concentrations that differ from other mammalian cells.

     
  15. 15.

    Semen samples were obtained from healthy donors with abstinence of at least 48 h. Data collection was carried out based on the principles outlined in the Declaration of Helsinki; all donors signed an informed consent agreeing to supply their own anonymous information and semen samples. The Ethics Committee of the Medical School, Universidad Nacional de Cuyo, approved the signed informed consent and the protocol for semen handling.

     
  16. 16.

    Occasionally, samples may not liquefy, making semen evaluation difficult. In these cases, mechanical mixing may be necessary. Inhomogeneity can be reduced by repeated (6–10 times) gentle passage through a blunt gauge 18 (internal diameter 0.84 mm) or gauge 19 (internal diameter 0.69 mm) needle attached to a syringe [36].

     
  17. 17.

    Evaluation of semen quality is done as a routine practice in our laboratory. Detailed protocols to assess semen quality are described on WHO laboratory manual for the examination and processing of human semen [36] and are beyond the scope of this chapter.

     
  18. 18.

    Spermatozoa are selected by their ability to swim out of seminal plasma and into culture medium, meaning that we obtain the motile fraction. This is known as the “swim-up” technique. The direct swim-up technique is the technique of choice when the semen samples are considered to be largely normal. This is our case, for that reason is the procedure chosen instead of discontinuous density gradients or simply washing [36]. The semen should preferably not be diluted and centrifuged prior to swim-up. Motile spermatozoa then swim into the culture medium.

     
  19. 19.

    Before removing an aliquot of semen for assessment, mix the sample well in the original container, but not so vigorously that air bubbles are created. This can be achieved by aspirating the sample ten times into a wide-bore (approximately 1.5-mm-diameter) disposable plastic pipette. Do not mix with a vortex mixer at high speed as this will damage the spermatozoa.

     
  20. 20.

    Assessment of sperm concentration. We generally use the Makler counting chamber (is only 10 μm deep: one-tenth of the depth of ordinary hemocytometers, making it the shallowest of known chambers). It contains two pieces of optically flat glass, the upper layer serves as a cover glass, with a 1-sq.mm fine grid in the center subdivided into 100 squares of 0.1 × 0.1 mm each. Spacing is firmly secured by four quartz pins [37].

    Counting Procedure:
    1. (a)

      Place a 10-μL drop of the immobilized, undiluted, and well-mixed preparation of liquefied semen at the center of the chamber by means of a pipette and cover it immediately. A microscopic objective of 20× is required.

       
    2. (b)

      Initiate the counting: the center of the chamber is subdivided into 100 squares of 0.1 × 0.1 mm each. Score sperm heads within ten squares in the same manner as blood cells are counted in a hemocytometer. The number obtained represents their concentration in millions per mL.

       
    3. (c)

      Rinse the chamber with water for reuse. Contact surfaces should be wiped with special lens paper after washing.

       
    4. (d)

      Alternatively, the Neubauer hemocytometer chamber can be used as described in the World Health Organization laboratory manual for the examination and processing of human semen [36].

       
     
  21. 21.
    Makler chamber is as accurate as the Neubauer chamber; therefore, either one can be used in routine semen analyses [38]. We describe here the method we currently use at the laboratory. Even though the results obtained with both chambers do not differ significantly, [39] the Makler chamber has some practical advantages over the Neubauer chamber:
    1. (a)

      Applied spermatozoa are uniformly distributed and monolayered and are observed in one-focal plane.

       
    2. (b)

      Dilution is unnecessary even with concentrated specimens. Analysis is carried out directly from original specimen in its natural environment.

       
    3. (c)

      The specimen can be analyzed quickly even by an inexperienced person.

       
     
  22. 22.

    After leaving the testis, mammalian spermatozoa are morphologically differentiated but are immotile and unable to fertilize. Progressive motility is acquired during epididymal transit. However, freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction and fertilizing an egg. They require a period of several hours in the female reproductive tract or in an appropriate medium in vitro to acquire this ability. The changes that occur during this period are collectively called capacitation [7]. This process is characterized by a special motility pattern and the ability to carry out the acrosome reaction under physiological stimuli [40, 41]. Many changes have been described during capacitation, including plasma membrane remodeling and flagellum hyperactivation. Major changes take place in the sperm plasma membrane, so as to render it fusogenic and responsive to ZP glycoproteins. However, the mechanisms involved have not been well defined. It is known to be a multistep process during which activation of a bicarbonate-dependent adenylyl cyclase leads to elevation of cAMP and protein kinase A (PKA)-mediated tyrosine phosphorylation of a subset of flagellar proteins that correlates with changes in sperm motility. Upstream of these events, determinable changes occur to the sperm’s plasma membrane, like membrane hyperpolarization, opening of voltage-gated Ca2+ channels, loss of transbilayer phospholipid asymmetry, and cholesterol efflux (for a review see Visconti [7]).

     
  23. 23.

    Loosen the cap of the tube to allow gas exchange. If a CO2 incubator is not available, use a HEPES-buffered medium, cap the tubes tightly and incubate at 37 °C.

     
  24. 24.

    Incubate the sperm with HTF alone to evaluate spontaneous acrosome reaction(background). To assess the maximum response, add only the stimulus (e.g., the calcium ionophore, A23187) and incubate for 15 min at 37 °C.

     
  25. 25.

    This step is crucial because if the BSA present in the spot is not removed, it will get stained by FITC-PSA, generating a background, which will make it impossible to distinguish the sperm staining pattern.

     
  26. 26.

    Important advances in the field of exocytosis have depended on the use of permeabilized cells that allow the composition of the cytosol to be precisely controlled. A number of techniques have been devised to create pores in plasma membranes, such that cells become leaky, but do not lyse. Many cell functions are controlled by molecular signals (hormones, neurotransmitters, and so forth) that interact with cell-surface receptors and trigger specific intracellular responses. Intracellular signaling can be difficult to study in isolated, purified systems, because these events depend on cellular architecture to a large extent. In intact cells, access to intracellular systems is limited by the restricted permeability of the plasma membrane [42, 43, 44, 45].

    Given that the sperm is a transduction and translationally inactive cell and that some reagents, proteins, and lipids required to dissect exocytosis molecular mechanisms are not permeant, a controlled plasma membrane permeabilization is necessary.

     
  27. 27.

    The units of SLO indicated are calculated for the batch of the protein we are using nowadays. Calculate your own units for your batch. The incubation performed in this step allows SLO binding to cholesterol molecules present in the plasma membrane.

     
  28. 28.

    Streptolysin O belongs to the homologous group of thiol-activated toxins that are elaborated by various Gram-positive bacteria. This toxin binds as monomers to cholesterol in the cytoplasmic membranes. They then oligomerize into ring-shaped structures, estimated to contain 50–80 subunits, which surround pores of approximately 30 nm diameter [46, 47].

     
  29. 29.

    Mix 10 μL of sperm dilution with 2 μL of eosin yellowish 0.5% in PBS on a slide placed on a 37 °C plate. After 1-min incubation, score cells in a conventional transillumination microscope with a 40× microscope objective.

     
  30. 30.

    Considering the hypothetical stock of 25,000 U/mL a diluted stock can be prepared to work. A 450-U/mL stock must be prepared as follows: mix 9 μL of 25,000 U/mL SLO, 100 μL of glycerol, and 1 μL of 5% BSA in PBS. Add PBS to a final volume of 500 μL. Aliquot and store at −20 °C if it can be used within a month; if not store at −80 °C.

     
  31. 31.

    The read out of our functional assays is the measurement of the acrosomal status after different treatments. Knowing whether the acrosomes have been exocytosed or not after the incubation with diverse reagents allows building a signal transduction network. The acrosomal status can be evaluated by different methods each with advantages and disadvantages (Table 1). The appropriate method should be chosen depending on the requirement. The techniques developed below could be used both for intact and permeabilized cells.

     
  32. 32.

    The protocol was adapted in our laboratory from the technique published by Larson and Miller [48]. This method is fast, easy, and inexpensive. It does not require a fluorescence microscope; a light optical microscope will suffice. Therefore, this technique can be used in ordinary diagnosis laboratories of biochemistry together with a routine semen analysis.

     
  33. 33.

    Prepare 10 mL of poly-l-Lysine solution by adding 9.5 μL of distilled water to 500 μL of 0.1% (w/v) poly-l-Lysine. Smear this solution over ethanol-cleaned slides. Let them dry.

     
  34. 34.
    View the slide under a light optical microscope at ×400 or ×600 magnifications. Categorize the spermatozoa as follows.
    1. (a)

      Acrosome-Intact: spermatozoa in which more than half the head is uniformly blue-stained (see Fig. 2).

       
    2. (b)

      Acrosome-Reacted: spermatozoa with light blue staining in the acrosome region (see Fig. 2).

       
    3. (c)

      Abnormal Acrosomes: all other spermatozoa. Perform scoring and classify the cells in the categories described.

       
     
  35. 35.

    Pisum sativum agglutinin has four subunits, two of approximately 17,000 daltons and two of about 6000 daltons. Lectin has specificity toward terminal α-d-mannosyl-containing oligosaccharides present in the acrosome granule. If the acrosome is present, the FITC-coupled lectin binds to the mannose residues and the structure shows fluorescent green staining. On the contrary, if the acrosome is lost, mannose residues are not present, and the lectin does not stain the granule.

     
  36. 36.

    The method is generally used to evaluate the acrosomal status by staining the sperm cells with FITC-coupled PSA, according to Mendoza et al. [49]. It is fast, easy, and yields accurate information about sperm exocytosis. The laboratory must be equipped with a fluorescence microscope or a flow cytometer (Table 1). Two procedures can be used for this staining: indirect or direct. In the indirect staining method, we first perform the functional assay as described in Subheading 3.4. Then, we fix/permeabilize the sperm and finally, we stain them. The spermatozoa that preserve the acrosome even after applying the stimulus are considered non-reacted or intact acrosome (Fig. 3, arrows, fluorescent acrosomes). On the contrary, if the spermatozoa lose the acrosomes, they are considered as reacted (Fig. 3, asterisk, no fluorescent acrosome).

    In the direct method, the lectin that binds the mannose residues of the acrosome is present in the media during the functional assay. Therefore, when the acrosome starts the exocytosis, the lectin enters through the pores, staining the granules of reacted cells. For this reason, fluorescent acrosomes are considered reacted and no fluorescent acrosomes are scored as intact.

    The difference between the two methods is that the direct method only detects the sperm reacting during the incubation. Any sperm that had reacted before (spontaneous acrosome reaction) is not stained by the lectin present in the medium [23].

     
  37. 37.

    Classification: a. Acrosome-Intact: spermatozoa in which more than half the head is brightly and uniformly fluorescent (Fig. 3). Acrosome-Reacted: spermatozoa with only a fluorescent band at the equatorial segment or no fluorescent stain at all in the acrosome region (Fig. 3).

     
  38. 38.

    Calculate acrosomal exocytosis indexes by subtracting the number of reacted spermatozoa in the negative control from all values and expressing the resulting values as a percentage of the acrosome reaction observed in the positive control. The average difference between positive and negative control must be around 15% or more (experiments where the difference between the negative and positive control is less than 10% must be discarded).

     
  39. 39.

    In the direct method, Pisum sativum agglutinin conjugated to FITC permeates into the acrosome when fusion pores open. The lectin stabilizes the acrosomal matrix preventing the dispersal of the granule contents [23].

     
  40. 40.

    It is very important to remove the PAF completely (cells cannot be run onto the cytometer if they are resuspended in PAF).

     
  41. 41.

    If it is not possible to run the cells on the cytometer right away, they can be stored up to 48 h at 4 °C, protected from light and resuspended in 1× PBS.

     
  42. 42.

    Another technique we would like to mention, given its importance, is the method published by Harper et al. [16] even though we do not use it as a routine practice. In this study, the progress of the AR is recorded in real time: fenestration of plasma membrane, exposure and dispersal of acrosomal content, and subsequent exposure of the IAM. A live human sperm is visualized using fluorescent labels for two different components of the acrosome. Soybean trypsin inhibitor (SBTI) binds to the acrosomal contents (specifically acrosin) of human sperm following membrane fenestration. The complement regulatory protein CD46 (membrane cofactor protein) is localized solely on the IAM in human sperm. As dispersal of the acrosomal content proceeds, binding sites for antiCD46 antibodies are revealed, allowing exposure of the IAM to be monitored. Simultaneous imaging of separate probes for acrosomal content and IAM show that rapid membrane fusion, initiated at the cell apex, is followed by exceptionally slow dispersal of acrosomal content (up to 12 min).

     
  43. 43.

    Induce the acrosome reaction with 10 μM A23187 or 15 μM Pg in intact cells or 10 μM calcium in permeabilized sperm (positive controls). Start to collect images once stimulus is added. We collect images in an Olympus FV1000 confocal microscope or an Eclipse TE300 Nikon microscope (2 frames/min) [27].

     
  44. 44.

    Perform background subtraction by selecting the region of interest placed as close as possible to the sperm of interest. Discard any incompletely adhered sperm that moved during the course of any experiment. Carry out fluorescence measurements in individual sperm by manually drawing a region of interest around the head of each cell. When required, express raw intensity values as relative fluorescence normalized to the maximum fluorescence obtained after the A23187 or calcium addition. Record the time of acrosome reaction initiation for each cell and calculate a cumulative frequency (number of reacted sperm at time t/number of reacted sperm after 1 h × 100).

     
  45. 45.

    For this technique, it is recommended to use a higher concentration of cells, at least 10 × 106 cells in 200 μL per condition. Refer to Subheading 3.4 and the same considerations must be taken for intact and permeabilized spermatozoa. Before processing the sample completely for TEM, perform a control of the functional assay by fluorescence microscopy as described in Subheadings 3.6.2 or 3.6.3. A complete revision of this technique can be found online http://www.bahiablanca-conicet.gob.ar/biblioteca/principios-practica-microscopia-electronica.pdf

     
Table 1

Comparison between acrosomal exocytosis assessment methods

Method

Advantage

Disadvantage

Method of choice

Coomassie blue staining method

Fast, easy, reliable, and inexpensive

Requires only optical light microscope

Can be used in fertility and biochemical laboratories for a routine semen analysis

Until now, the method has not been set up for flow cytometry; that would allow an accurate counting independent of the human eye

Only to assess sperm exocytosis

Fluorescence assessment of acrosomal status. Pisum sativum Agglutinin coupled to fluorescein isothiocyanate (FITC-PSA)

Direct and indirect staining

Fast, easy, and reliable

Allows an accurate counting by flow cytometry, widely used

Permits measurement of the exocytosis in real time (kinetic studies)

More expensive; due that a fluorescence microscope equipment is needed or a flow cytometer

It is not used routinely in diagnostic laboratories

If you do not want to detect spontaneous acrosome reaction occurring before applying the stimulus, you should use the direct method. It only detects sperm reacting during the incubation. Any sperm that had reacted before is not stained by the lectin present in the medium [23]

Real-time measurements of exocytosis require the use of the direct method [27]

Acrosome reaction assessment by transmission-electron microscopy

Allows the observation of the sequence of events that occurs in a spermatozoa’s head during human sperm acrosome reaction. Particularly, the acrosome morphology at the ultrastructural level

It requires a week to be finished

Experienced and trained professionals

If you need to study the ultrastructure of the exocytosis, this is the method of choice

You can score and analyze different changes of the granule like swelling, membrane curvature after different treatments, number and depth of invagination of the OAM, etc

Fig. 4

Measurement of the AR by a direct method using flow cytometry. Dot plot graphs of a representative experiment showing the number of FITC-PSA stained cells. Ten thousand cells were counted on each event and are represented by a single dot (side scatter (SSC) vs. FITC-PSA fluorescence). Note the low fluorescence index in control population (a) without any stimulus compared to the increased fluorescence that appeared upon incubation with a stimulus (b)

Fig. 5

Electron micrographs show the ultrastructural morphological correlate represented in the cartoons in Fig. 1. (a) Two cells classified as intact (unreacted) sperm (arrowheads). Both sperm heads show intact acrosomes (a), nucleus (n), and equatorial segment (es). (b) Swollen acrosome after stimulation (as). Invaginations of the outer acrosomal membrane contribute to the formation of the limiting surface of future hybrid vesicles (hv). Notice the close membrane apposition (ma) between the plasma and the outer acrosomal membranes at the edge of the invaginations. Some vesicles inside the acrosome (asterisk). (c) Micrograph showing sperm with a vesiculated acrosome. The membranes at the edge of invaginations detached from the head, and hybrid vesicles (hv) are released. (d) A sperm that has completed the acrosome reaction. The equatorial segment (es) remains unaltered during the vesiculation process (es)

Notes

Acknowledgments

The authors thank E. Bocanegra and R. Militello for technical assistance. We specially thank the excellent contribution of P. López, MS from the STAN: ST3371 of TEM and SEM samples preparation, IHEM-CONICET-UNCuyo.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Laila Suhaiman
    • 1
  • Karina Noel Altamirano
    • 2
  • Alfonsina Morales
    • 2
  • Silvia Alejandra Belmonte
    • 2
  1. 1.Instituto Interdisciplinario de Ciencias Básicas (ICB) CONICET. Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de CuyoMendozaArgentina
  2. 2.Instituto de Histología y Embriología de Mendoza (IHEM) “Dr. Mario H. Burgos”. CONICET. Facultad de Ciencias MédicasUniversidad Nacional de CuyoMendozaArgentina

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