Abstract
The acrosome reaction (AR) is an essential process of spermatozoa for fertilization and is characterized by the exocytosis of the acrosomal content and the release of hybrid membrane vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. Through acomplex membrane fusion process, which exposes the inner acrosomal membrane, both the morphology of the plasma membrane and the distribution of proteins involved in sperm–egg interaction are changed [1, 2]. AR also modifies sperm function, since acrosome-reacted sperm are no longer able to bind to the zona pellucida, but to the oolemma [3].
9.1 Introduction
The acrosome reaction (AR) is an essential process of spermatozoa for fertilization and is characterized by the exocytosis of the acrosomal content and the release of hybrid membrane vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. Through acomplex membrane fusion process, which exposes the inner acrosomal membrane, both the morphology of the plasma membrane and the distribution of proteins involved in sperm–egg interaction are changed [1, 2]. AR also modifies sperm function, since acrosome-reacted sperm are no longer able to bind to the zona pellucida, but to the oolemma [3].
Given the biological importance of the acrosome reaction, several techniques to assess this exocytotic process have been developed. Determination of the ability of spermatozoa to undergo the acrosome reaction has turned out to be a useful parameter in order to evaluate male infertility [4]. The difference between spontaneous and induced acrosome reaction, i.e. the inducibility of acrosome reaction, is of prognostic value for the sperm fertilizing capacity [5]. For diagnostic purposes, it is important that the results from different laboratories are comparable. This is only possible if the methods equally detect acrosomal events and result in comparable percentages of acrosome reaction. Dyes and fluorescein-conjugated lectins bind to different structures of the acrosome [6]. Staining protocols and assays based on lectins labeled with fluorescent dyes are easy to perform, relatively inexpensive and frequently used to quantify sperm acrosome reaction [7]. More sophisticated methods such as immune cytochemistry with monoclonal antibodies or electron microscopy are not available in all laboratories [8]. Recently, the incorporation of flow cytometry into these protocols has led to new, easy, reproducible, reliable and fast protocols for the determination of this important sperm function [9].
9.1.1 Sperm Function
Prior to fertilization, spermatozoa must undergo a series of physiological changes before being able to fertilize an oocyte. These changes occur in the female reproductive tract, where spermatozoa interact with the microenvironments in the female genital tract during their journey to meet the oocyte in the ampulla of the fallopian tube. Finally, the spermatozoon binds to the oocyte, triggers acrosome reaction and hypermobility, a movement that facilitates the penetration of oocytes’ covering [2].
The support of this structure is obtained by the cytoskeleton, constituting the plasma membranes and acrosome membranes. The main element of the cytoskeleton of the sperm head is the perinuclear theca, a rigid capsule that covers the nucleus and that once the fusion of plasmatic membrane and outer acrosome membranes occurs with the acrosome reaction, must preserve the integrity of the DNA.
The equatorial segment corresponds to the area where the inner and outer acrosomal membranes converge and fuse, and contains the receptors involved in the sperm binding. The post acrosome sheath is associated with the oocyte activation [10, 11].
9.1.2 Acrosome Reaction
This species-specific process implies the existence of molecules for recognition between male and female gametes and is triggered by the presence of oviductal fluid factors that aid in extracellular signaling and recognition of the oocyte [12, 13]. The AR can occur spontaneously and can also be induced in vitro. It is indispensable for the fertilization process and is generated as a result of the action of suitable inductors (reviewed by [8]).
The acrosome is derived from the Golgi apparatus during spermiogenesis and because of its origin, structure and cellular function, it is comparable to a lysosome. Unlike normal lysosomal exocytosis, acrosome reaction is regulated by a complex mechanism involving phosphoinositide, interaction of endogenous and exogenous nucleotides, Ca2+, calmodulin, microtubules and microfilaments. In the initial steps of acrosome reaction, outer acrosomal membrane and plasma membrane fuse at various sites forming so-called hybrid-membrane vesicles, a process leading to a fenestration of the acrosome with eventual complete acrosomal loss and the release of the acrosomal contents. As a result, instead of exposing the outer acrosomal membrane, the internal acrosomal membrane is exposed as a new rostral surface membrane. The vesicles release their content of hydrolytic enzymes, as the sperm penetrates through the zona pellucida, allowing the spermatozoon to bind to the oolemma and eventually enter the oocyte.
In vivo, the acrosomal reaction begins with the stimulation of a natural agonist such as progesterone or the zona pellucida. This stimulus causes an entry of Ca2+ into the spermatozoa, which involves ATPases whose function is to maintain low levels of Na+ and Ca2+ and high levels of K+. The entry of Ca2+ causes the deactivation of ATPases, in addition to an increase in intracellular Na+ with a H+ output and consequently, an increase in intra-acrosomal pH, which induces the activation of enzymes such as acrosin. The increase in intracellular Ca2+ induces the formation of second messengers, which are incorporated into a cascade of events that occurs during the acrosome reaction and the activation of enzymes such as phospholipase A2 generating lysophosphatidylcholine and arachidonic acid that are both necessary for the fusion of membranes needed in the acrosomal reaction (review by Beltrán et al., [14]). The specific recognition of gametes, the sperm binding with its acrosome intact to zona pellucida, the induction of the acrosomal reaction and the prevention of polyspermia are related to the zona pellucida protein ZP3. ZP3 is the protein involved in the sperm binding and induction of acrosome reaction. This process is referred to as primary binding. Subsequently, the sperm can start penetrating the zona and interact with another zona protein, ZP2 [15]. This binding is stronger and is referred to as secondary binding [16].
9.1.3 Factors to Induce Acrosome Reaction
Acrosome reaction can be induced under artificial conditions (in vitro) with Test-Yolk buffer, albumin addition, zona pellucida, follicular fluid, calcium ionophore, prolonged incubation times, low incubation temperatures and hypertonic media among others [17, 18]. The determination of the ability of spermatozoa to perform AR is used as a test to evaluate sperm function since it correlates with the results of in vitro fertilization [8]. The three most commonly used inducers of AR are (a) low temperature, (b) calcium ionophore and (c) progesterone, with the latter inducer being considered physiological.
9.1.3.1 Low Temperature
The AR is induced by low temperature according to Sanchez et al [19]. After swim-up, sperm suspensions are treated in two different ways: (i) Control group: 24 hours at room temperature, additional incubation at 37°C for 3 hours; (ii) Experimental low temperature: 24 hours at 4°C, additional incubation at 37°C for 3 hours. Following the experimental procedures, slides with spermatozoa are prepared to determine the acrosomal status of spermatozoa. This method correlates well with in vitro fertilization, and a total AR <13 percent and inducibility of AR <7.5 percent is associated with low number of oocyte fecundation in in vitro fertilization [5].
9.1.3.2 Calcium Lonophore
The AR is induced by calcium ionophore according to Ozaki et al. [20]. A stock solution of calcium ionophore A23187 (9.55 × 10−3 M in 100 percent DMSO) is prepared and stored at −20°C. Working solutions are prepared by diluting the thawed stock of ionophore 1:10 in modified human tubular fluid 30 minutes before stimulation of the AR. Aliquots of the working solutions are added to the samples to achieve concentrations of 10 μM ionophore (0.1 percent v/v DMSO). Control samples are treated simultaneously under the same conditions, except that only 0.1 percent DMSO is added without ionophore. An infertile male shows a significantly reduced number of acrosome-reacted spermatozoa [21]. The difference between complete acrosome-reacted spermatozoa with and without treatment is determined. A percentage difference of ≤5 percent indicates male infertility [22].
9.1.3.3 Progesterone
Spermatozoa (107 cells/mL) are prepared in Biggers, Whitter and Whittingham medium (BWW, pH 7.2; Genmed Scientifics Inc., USA) and incubated for 3 hours at 37°C and 0.5 percent CO2 (sperm viability is more than 95 percent after incubation). Afterwards, an aliquot of capacitated spermatozoa is incubated with progesterone (P4; final concentration of 40 µM) according to Chen et al. [23]. The P4‐induced AR should be more than 24 percent and is significantly correlated to good in vitro fertilization rate [23, 24].
9.1.4 Tests Used to Measure Acrosome Reaction
Several techniques have been proposed to differentiate acrosome-intact from acrosome-reacted human sperm with different staining techniques; light and electron microscopic examination, indirect immunofluorescence using polyclonal and monoclonal antibodies and labeling with fluorescein-conjugated lectins.
9.1.4.1 Direct Tests
The direct method to evaluate the AR is transmission electron microscopy. Considered the most accurate to detect the AR, both for clinical practice and for research, its use is limited, especially due to the requirement of very costly equipment and specialized personnel for the preparations and analysis. It is also a laborious method and determines only a limited number of cells.
9.1.4.2 Indirect Tests
These tests are the most commonly used and can be based on histochemical methods with different stains using light microscopy, such as the triple stain technique or antibodies and lectins that are labeled with fluorescent molecules that can be determined with immunofluorescence microscopy or flow cytometry. Since fluorescence techniques are more cited protocols, these techniques have been selected for discussion in this chapter.
9.2 Fluorescence Detection of the Acrosome
9.2.1 Principle of Hoechst 33258/FITC-Pisum sativum Agglutinin (PSA-FITC) Staining in Simultaneous Assessment of Viability and Acrosome Reaction Rate of Human Sperm
9.2.1.1 Principle
The PSA-FITC lectin is a dimeric lectin, consisting of two ab monomers with high affinity for α-D-mannose and α-D-glucose residues of glycoproteins present in the plasma membrane of the acrosomal domain of the sperm head. This technique is a negative staining technique that stains the acrosomal area of acrosome-reacted sperm, thus those sperm that lost their acrosome [25]. The PSA-FITC has been extensively used to assess AR in fixed human sperm [20].
9.2.1.2 Protocol
Supravital nuclear staining with bis-benzimide (Hoechst 33258; Sigma) is used to identify dead sperm cells. The dye is added to the sperm suspensions (approximately 1 × 106 spermatozoa) one minute before the final washing prior to preparation of the sperm smear. To permeabilize the sperm membrane, the slides are treated with 100 percent methanol for 30 seconds, followed by incubation for 30 minutes in a moisture chamber at room temperature in phosphate-buffered salt (PBS) containing 50 mg/mL Pisum sativum agglutinin conjugated with fluorescein isothiocyanate, pH 7.4, (FITC-PSA; Sigma) according to [26]. The smears are washed for at least 15 minutes in distilled water, air-dried and examined under an epifluorescence microscope (excitation filter 450–490 nm, dichroic mirror 510 nm, barrier filter 520 nm). For assessment of viability and acrosomal status, 200 spermatozoa are examined in randomly selected fields according to the fluorescence pattern of their acrosomes.
9.2.1.3 Interpretation of Results
The following criteria are used to evaluate the acrosomal status and viability of spermatozoa.
(i) A spermatozoon showing the region of the nucleus marked with Hoechst 33258 (blue) is considered dead.
(ii) A spermatozoon showing the acrosomal region totally or partly marked with PSA-FITC (yellow-green) is considered to be acrosome-reacted.
(iii) Live spermatozoa showing more than 2/3 of the acrosome with fluorescence are considered acrosome-reacted.
9.2.2 Principle of FITC-Concanavalin A (FITC-ConA) Staining in Simultaneous Assessment of Viability and Acrosome Reaction Rate of Human Sperm
9.2.2.1 Principle
Concanavalin A binds specifically to α-mannose and α-glucose residues on the inner acrosomal membrane of the head area of human sperm that will be exposed in only acrosome-reacted sperm [27].
9.2.2.2 Protocol
Supravital nuclear staining is determined with Hoechst 33258 (stock solution: 1 mg/mL) dissolved in Dulbecco buffer phosphate, added to a final concentration of 1 μg/mL and co-incubated with sperm for 10 minutes. The samples are centrifuged twice at 300 × g for 10 minutes and the pellet resuspended in 500 μL of human tubular fluid medium. Spermatozoa are fixed in 10 percent formalin solution (neutral buffered) for at least 60 minutes at room temperature and centrifuged at 300 × g for 10 minutes. Most of the supernatant is removed, and the sperm pellet is resuspended in the remaining solution. A 200 μL aliquot of the fixed sperm suspension is smeared and air-dried on a glass slide coated with 0.05 percent poly-L-lysin solution (5 μL). The slide is rinsed with 0.2 M glycine solution and washed with 10 mM PBS. Spermatozoa are labeled with 25 μL of FITC-ConA 100 μg/mL in 10 mM PBS, pH 7.4) for 25 minutes at room temperature according to Holden et al. [27].
Afterwards, the slides are rinsed with an excess of 10 mM PBS and mounted. At least 200 sperm are examined and scored per slide using a fluorescence microscope (magnification is 1000×).
9.2.2.3 Interpretation of Results
The following criteria are used to evaluate the acrosomal status.
(i) Sperm with positive fluorescence in the acrosomal region are scored as acrosome-reacted.
(ii) Sperm without fluorescence in the acrosomal region are considered as acrosome-intact.
(iii) A spermatozoon showing the region of the nucleus marked with Hoechst 33258 (blue) is considered dead.
9.2.3 Principle for FITC-Peanut Agglutinin (FITC-PNA) Staining in Simultaneous Assessment of Viability and Acrosome Reaction Rate of Human Sperm
9.2.3.1 Principle
FITC-PNA binds specifically to the outer acrosomal membrane. In sperm with an intact acrosome, the acrosomal region of the sperm head exhibits a uniform apple-green fluorescence. In acrosome-reacted sperm, only the equatorial segment of the acrosome is stained [28]. This is a method used in our laboratory (Figure 9.1).
