Molecular Human Reproduction, Vol. 8, No. 8, 722-727,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive Gi protein and extracellular calcium, and priming effect of progesterone and follicular fluid
1 The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia, USA, 2 Androlab-Reproductive Medicine and Andrology Clinic, Curitiba, Brazil and 3 Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Stellenbosch, Cape Town, Republic of South Africa
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Abstract |
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In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on Gi protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA–FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 µg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of Gi proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.
acrosome reaction/calcium/follicular fluid/G protein/human sperm/progesterone/solubilized zona pellucida
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Introduction |
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The acrosome reaction is a prerequisite for fertilization in mammalian sperm (Yanagimachi, 1994
). In the mouse, one of the best characterized species so far, acrosomal exocytosis is physiologically induced by components of the zona pellucida (ZP), particularly ZP protein 3 (ZP3) (Bleil and Wassarman, 1980, 1983; Florman and Storey, 1982; Florman and Wassarman, 1985; Ward et al., 1992). Binding of ZP3 to putative complementary receptor(s) on the sperm surface activates transmembrane signals that trigger cellular cascades resulting in the acrosome reaction (Wassarman, 1990a,b, 1999; Saling, 1991).
Several cellular pathways are involved in the stimulation of the acrosome reaction. It has been demonstrated that activation of pertussis toxin-sensitive heterotrimeric G proteins (Gi class) is necessary for the ZP-induced acrosome reaction in the murine model (Kopf et al., 1986; Kopf, 1990). Gi protein acts as a signal transducing element downstream of ZP3–receptor interactions and couples receptor occupancy to changes in ionic conductance and/or a variety of intracellular second messenger cascade systems whose activation in turn results in release of acrosomal contents (Kopf, 1990). One of such elements is likely to be a pH regulator, resulting in a transient alkanization of intracellular pH (Florman et al., 1989, 1998; Kopf, 1990). Second messengers include the adenylate cyclase–cAMP system resulting in activation of protein kinase A (PKA) leading to phosphorylation of specific, putative proteins resulting in exocytosis. In addition, the activation of phospholipase C (PLC) may lead to 1,2 diacylglycerol (DAG) and inositol 1,4-5 trisphosphate (IP3) formation. DAG may stimulate protein phosphorylation through protein kinase C (PKC), whereas IP3 may activate intracellular calcium release through modulation of IP3-sensitive intracellular calcium stores (Kopf, 1990; Florman et al., 1998; Wassarman, 1999).
It has also been proposed that the ZP may alternatively activate a low voltage- activated T type calcium channel that is pertussis toxin-insensitive (Florman et al., 1992, 1998; O'Toole et al., 2000). Activation of pertussis toxin-sensitive and -insensitive mechanisms leads to significant and sustained changes in intracellular calcium levels, a prerequisite for the acrosome reaction (Kopf, 1990; Florman et al., 1998).
Progesterone, present in high concentrations in the follicular fluid, is also a known stimulator of the acrosome reaction. It has been shown that progesterone exerts a priming effect on the ZP-stimulated acrosome reaction in the mouse (Roldan et al., 1994). In such studies, treatment with progesterone followed by ZP led to maximal generation of DAG and maximal breakdown of phosphatidylinositol-4,5-bis-phosphate, signalling a priming role for progesterone in the initiation of exocytosis.
Relatively few studies have addressed the role of the physiological, homologous inducer of the acrosome reaction, the ZP, in human sperm. This is probably due to the difficulty in obtaining human material (oocytes) to perform such experiments. ZP can be obtained from oocytes recovered from ovarian tissue (post-surgical or post-mortem) or from IVF treatment following appropriate patient consent for donation.
Cross et al. were the first to report that treatment of human sperm in suspension with acid-disaggregated human ZP (2–4 ZP/µl) increased the incidence of acrosome-reacted sperm (Cross et al., 1988). Lee et al. demonstrated that pertussis toxin treatment of human sperm inhibits the (solubilized) ZP-induced acrosome reaction (Lee et al., 1992). In contrast, acrosomal exocytosis induced by the calcium ionophore A-23187 is not inhibited by pertussis toxin pretreatment. Studies by Franken et al. showed a dose-dependent effect of solubilized human ZP on the acrosome reaction in the range of 0.25–1 ZP/µl and also confirmed the involvement of Gi protein during ZP-induced acrosome reaction of human sperm (Franken et al., 1996).
Capacitated human sperm also respond to a progesterone stimulus in vitro by a rapid increase in intracellular free calcium due to the promotion of calcium influx (Thomas and Meizel, 1989; Blackmore et al., 1990, 1991). Progesterone activates a calcium channel that has yet to be defined at the molecular level in the human (Blackmore and Eisoldt, 1999). Recent findings have revealed the molecular structure of such an ion channel in murine species (Ren et al., 2001). The entire increase in intracellular calcium levels induced by progesterone is abolished when the extracellular calcium is removed by the addition of the calcium chelator EGTA to the extracellular medium (Blackmore et al., 1990). There is evidence for both L- and T-type calcium channels in mouse and human sperm (Blackmore et al., 1990, 1991; Benoff et al., 1994; Arnoult et al., 1996 Arnoult et al., 1997; Shiomi et al., 1996; Florman et al., 1998; Blackmore and Eisoldt, 1999). It has been proposed that progesterone reacts with a multireceptor system on the sperm surface and that this system co-operates with that used by the ZP to control the physiological acrosome reaction (Mendoza et al., 1995; ESHRE Andrology Special Interest Group, 1996). In human sperm, progesterone effects are not associated with Gi protein activation (Tesarik et al., 1993).
The first signal transduction–second messenger pathway demonstrated to have a role in human sperm acrosome reaction involved the cAMP/PKA system (De Jonge et al., 1991a). The PLC-PKC system was also demonstrated to play a role in human sperm exocytosis (De Jonge et al., 1991b; Bielfeld et al., 1994; Doherty et al., 1995). However, it is unclear which of such mechanisms is the most significant under physiological conditions and how the various systems cross-talk.
Although transmission electron microscopy still represents the gold standard for the evaluation of acrosomal status, this method is expensive and laborious and therefore it cannot be used routinely (Zaneveld et al., 1991; Kohn et al., 1997). Other well established methods are currently being used to assess the acrosome reaction in humans. The most widely used method involves lectins [i.e. Pisum Sativum agglutinin (PSA) and others] labelled with fluorescence [i.e. fluorescein isothiocynate (FITC)]. The inducibility of the acrosome reaction following calcium ionophore challenge using PSA–FITC has been recommended as the presently available optimal method to assess this physiological event under in-vitro conditions in human sperm (Tesarik, 1985, 1989; Cummins et al., 1991; ESHRE Andrology Special Interest Group, 1996; Kohn et al., 1997; Oehninger et al., 2000).
Franken et al. reported the validation of a new microassay using minimal volumes of solubilized, human ZP to test the physiological induction of the acrosome reaction in human sperm (Franken et al., 2000). In such studies, a dose-dependent effect of solubilized ZP on acrosomal exocytosis was observed reaching maximal induction using 1.25–2.5 ZP/µl for both the microassay and the standard (macro) assay. Furthermore, the inducibility of the acrosome reaction by a calcium ionophore was similar in both assays.
Here, we aimed to further characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the ZP and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the ZP-induced acrosome reaction on Gi protein activation and the presence of extracellular calcium; and (ii) to determine whether progesterone or follicular fluid exert a priming or synergistic effect on the ZP-induced acrosome reaction. Purified populations of highly motile sperm were recovered from the ejaculates of fertile donors and exposed to the agonists in a microvolume assay. Acrosomal exocytosis of live sperm was determined with indirect immunofluorescence using PSA–FITC/Hoechst double-staining.
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Preparation of sperm samples
Ejaculates from fertile men participating in our artificial insemination donor programme were used in these studies. Approval was obtained from the Institutional Review Board of Eastern Virginia Medical School, where most experiments were performed. Sperm concentration and percentage progressive motility were objectively evaluated using the HTM-IVOS semen analyser version GS 771 (Hamilton Thorne Research, Beverly, MA, USA) with fixed parameter settings (Oehninger, 1995
). Sperm concentration and motility readings were manually monitored and corrections were made as appropriate. Sperm concentration and motility were assessed according to the World Health Organization criteria (World Health Organization, 1999) and sperm morphology was examined according to strict criteria after Diff-Quik staining (Kruger et al., 1986). The sperm fractions with high motility were isolated by discontinuous Percoll (Sigma Chemical Co., St Louis, MO, USA) gradient separation (90 and 40% layers) using human tubal fluid (HTF; Irvine Scientific, Santa Anna, CA, USA) as diluent. Up to 2 ml of semen was carefully placed on Percoll layers, centrifuged at 380 g for 20 min and the pellet of the 90% layer was mixed with HTF and then centrifuged at 380 g for 10 min. The supernatant was discarded and the pellet was resuspended to achieve a sperm concentration of 10x106/ml.
The original sperm parameters of the samples used were as follows (mean ± SD): concentration 101 ± 10x106/ml; sperm motility 60 ± 5%, and normal morphology (strict criteria) 15 ± 3%. The motile sperm fractions recovered from the 90% Percoll layers (10x106 cells/ml, >90% motility) were allowed to capacitate for 3 h at 37°C under 5% CO2 in water-saturated air in HTF supplemented with 3% human serum albumin (HSA; Irvine).
Preparation of solubilized ZP
Human oocytes were retrieved from post-mortem derived ovarian tissue following approval by the local ethics committee at Stellenbosch University. Oocytes were stored in HTF using DMSO/sucrose at –196°C in liquid nitrogen (Franken et al., 1989). Oocytes were shipped to Norfolk and 12 h prior to each experiment were removed from storage and thawed at 37°C. The oocytes were placed in 0.25 mol/l sucrose and 3% HSA in modified HTF medium for 20 min at room temperature, after which they were placed under mineral oil (Sigma) until use.
Prior to each experiment, oocytes were vigorously pipetted with a small-bore glass pipette (inner diameter 80 microns) to separate the ZP from the ooplasm. The separated ZPs were then placed in a plastic Eppendorf tube containing modified HTF medium supplemented with 3% HSA. The tubes were centrifuged for 15 min at 1800 g, after which the HTF medium was carefully removed under microscopic vision, leaving only the ZP at the bottom of the tube. Solubilization of the ZP was performed in a microvolume under microscopic control following addition of 10 mmol/l HCl; after this, 10 mmol/l NaOH was added to the zona to render a final ZP concentration of 2.5 ZP/µl. The final pH of the ZP solution was 7.2–7.4 (Franken et al., 1996, 2000).
Calcium ionophore
The calcium ionophore A23187 (Sigma) was prepared in a stock solution with dimethylsulphoxide (Sigma) and then diluted in modified HTF to be tested at a final concentration of 5 µmol/l in the acrosome reaction microassay (Franken et al., 2000).
Progesterone
Progesterone (Sigma) was prepared in a stock solution with ethanol and then diluted with phosphate-buffered saline (PBS) to be tested at a final concentration of 1.25 µg/ml (Sueldo et al., 1993; Oehninger et al., 1994).
Human follicular fluid
A pool of follicular fluid was obtained from women participating in our IVF programme and receiving gonadotrophin stimulation, after obtaining approval from the Institutional Review Board. Individual fluids were used following oocyte identification and only fluids from follicles containing a mature metaphase II oocyte were studied. Each tube of follicular fluid was centrifuged at 4°C for 15 min at 1500 g and stored at –20°C until use (Marin-Briggiler et al., 1999). For the acrosome reaction, the follicular fluid was tested at a final concentration of 10% in PBS.
Acrosome reaction
Assessment of the acrosome reaction was performed using a microassay as previously described (Franken et al., 2000) and modified as described below. Briefly, 1 µl of ZP solution (concentration 2.5 ZP/µl) (or 1 µl of the agonists A23187, progesterone or human follicular fluid) was aspirated into a Teflon pipette tip (Hamilton Pipette-tip; Separations, Cape Town, South Africa), fitted to a microsyringe (Hamilton 702; Separations) with 1 µl of sperm, to render a final ZP concentration of 1.25 ZP/µl (or 5 µmol/l A23187, 1.25 µg/ml of progesterone or 10% human follicular fluid). Prior to aspiration into Teflon tips, all sperm/ZP suspensions were gently mixed in a well of a microtitre plate (Laboratory and Scientific, Cape Town, South Africa). To prevent evaporation from the Teflon tips, HTF was aspirated into both sides of the Teflon tip to seal off sperm–ZP suspensions. Each sperm–ZP suspension was separated from the HTF droplets by air bubbles on both sides.
Control and treated sperm samples were carefully removed from the Teflon tips and placed on separate spots on the spotted slide and immediately evaluated for motility under an inverted phase contrast microscope (Nikon, Garden City, NY, USA). The percentage of live cells was recorded by adding 1 µl (0.3 µg/ml) Hoechst-dye (Bis-Benzimide supravital stain, Hoechst 33258, B-2883; Sigma) to each spot for 5 min.
Following motility assessment, the sperm droplets were allowed to air dry, fixed in 70% ethanol for 20 min, and then simultaneously evaluated for percentage live cells and acrosomal status by Hoechst/FITC–PSA (Sigma) with epifluorescence microscopy at a magnification of x1000 using a phase-contrast microscope (Eclipse 600; Nikon, Melville, NY, USA) equipped with a digital camera with a high pressure mercury lamp power supply (SPOT RT, software version 3.2; Diagnostic Instruments, Augusta, GA, USA). Two hundred cells were counted in a blinded fashion in each well of the spotted slide and results were expressed as percentage live, acrosome-reacted sperm. The following staining patterns were evaluated as acrosome reacted sperm: (i) distinct staining in the equatorial region occurring as an equatorial bar; (ii) no staining observed over entire sperm surface; and (iii) patchy staining on acrosomal region (Cross et al., 1988; Cummins et al., 1991; Mahony et al., 1991; Franken et al., 1996; 2000).
Experimental design
In the first experiment (experiment 1), we compared the acrosome reaction-inducing ability of solubilized ZP and the calcium ionophore, and also examined the impact of inactivation of G protein by pertussis toxin and calcium chelating from the extracellular medium by EGTA. An ejaculate from each of five different donors was subjected to a separation of the motile fraction followed by a 3 h capacitation period as described above. Each sample was then aliquoted into five parts and incubated under the following different conditions at 37°C under 5% CO2 in water-saturated air: (i) control medium (HTF supplemented with 3% HSA for 60 min); (ii) calcium ionophore at a final concentration of 5 µmol/l for 60 min; (iii) solubilized ZP at a final concentration of 1.25 ZP/µl for 60 min; (iv) pretreatment with the calcium chelator EGTA [ethylene (oxyethylenenitrilo) tetra-acetic acid; Sigma] at a final concentration of 5 µmol/l for 30 min, followed by solubilized ZP at a final concentration of 1.25 ZP/µl for 60 min; and (v) pretreatment with the functional inactivator of G protein, pertussis toxin (Calbiochem, San Diego, CA, USA) at a final concentration of 100 ng/ml for 30 min, followed by solubilized ZP at a final concentration of 1.25 ZP/µl for 60 min. After incubation, each condition was tested for the percentage of live, acrosome-reacted sperm in the microassay as detailed above.
In the second experiment (experiment 2), we compared the acrosome reaction-inducing ability of solubilized ZP and progesterone, and also examined the impact of sequential treatment of ZP and progesterone and in reversed order. Twenty-seven ejaculates from five different donors were subjected to a separation of the motile fraction followed by a 3 h capacitation period as described above. Each sample was then aliquoted into five parts and incubated under the following different conditions at 37°C under 5% CO2 in water-saturated air: (i) control medium (HTF supplemented with 3% HSA) for 30 min; (ii) solubilized ZP at a final concentration of 0.5 ZP/µl for 30 min; (iii) progesterone at a final concentration of 1.25 µg/ml for 30 min; (iv) solubilized ZP at a final concentration of 0.5 ZP/µl for 15 min followed by progesterone at a final concentration of 1.25 µg/ml for an additional 30 min; and (v) progesterone at a final concentration of 1.25 µg/ml for 15 min followed by solubilized ZP at a final concentration of 0.5 ZP/µl for 30 min. After incubation, each condition was tested for the percentage of live, acrosome-reacted sperm in the microassay as detailed above.
In the third experiment (experiment 3a), we compared the acrosome reaction-inducing ability of solubilized ZP and follicular fluid, and also examined the impact of sequential treatment with follicular fluid followed by solubilized ZP. An ejaculate from each of three different donors was subjected to a separation of the motile fraction followed by a 3 h capacitation period as described above. Each sample was then aliquoted into five parts and incubated under the following different conditions at 37°C under 5% CO2 in water-saturated air: (i) control medium (HTF supplemented with 3% HSA) for 60 min; (ii) calcium ionophore at a final concentration of 5 µmol/l for 60 min; (iii) solubilized ZP at a final concentration of 1.25 ZP/µl for 60 min; (iv) human follicular fluid (10%) for 60 min; and (v) pretreatment with human follicular fluid at 10% for 30 min followed by solubilized ZP at a final concentration of 1.25 ZP/µl for 60 min. After incubation, each condition was tested for the percentage of live, acrosome-reacted sperm.
In a subsequent follow-up experiment (experiment 3b), we examined the kinetics (time dependency) of the acrosome reaction-inducing ability of solubilized ZP following pretreatment with follicular fluid. An ejaculate from each of three different donors was subjected to a separation of the motile fraction followed of a 3 h capacitation period as described above. Each sample was then incubated for 30 min with human follicular fluid (10%) at 37°C under 5% CO2 in water-saturated air. Thereafter, the sample was aliquoted into four parts and further incubated with human solubilized ZP (final concentration of 1.25 ZP/µl) for four different time periods: 15, 30, 45 and 60 min. An aliquot exposed only to follicular fluid under the same conditions served as a control. After incubation, each condition was tested for the percentage of live, acrosome-reacted sperm.
Statistical analysis
Comparisons of the percentage of live, acrosome-reacted sperm under the different experimental conditions were performed using one-way or repeated measures analysis of variance (ANOVA) as appropriate. The Tukey–Kramer multiple comparisons test was used to assess individual differences among conditions tested. P-values < 0.05 were considered significant. Results are expressed as mean ± SE.
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The results of experiment 1 are depicted in Figure 1
. The overall results analysed by ANOVA had a significance of P < 0.0001. The calcium ionophore A23187 significantly increased the acrosome reaction from 12 ± 2% (control conditions) to 29 ± 3% (P < 0.001). The solubilized ZP produced an increase in acrosomal exocytosis (49 ± 5%) that was significant higher than control and calcium ionophore treatment conditions (P < 0.001 versus both conditions). Pretreatment with EGTA followed by ZP resulted in a significantly lower rate of acrosome reaction (22 ± 2%) than ZP alone (P < 0.001), but this percentage of acrosomal exocytosis was significantly higher than control conditions (P < 0.01). Pretreatment with pertussis toxin followed by ZP also resulted in a significantly lower rate of acrosome reaction (25 ± 3%) than ZP alone (P < 0.001), but this percentage of acrosomal exocytosis was also significantly higher than control conditions (P < 0.001). The results of this experiment demonstrated that: (i) the calcium ionophore and solubilized ZP are powerful stimulators of acrosomal exocytosis under microassay conditions; and (ii) the functional inactivation of Gi proteins (pertussis toxin-sensitive) and the absence of extracellular calcium are capable of inhibiting the solubilized ZP-induced acrosome reaction.
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Figure 2
shows the results of experiment 2. The overall results analysed by ANOVA had a significance of P < 0.0001. Both progesterone (27 ± 3%) and ZP (28 ± 2%) resulted in a significant enhancement of the acrosome reaction when compared with control conditions (12 ± 1%; P < 0.001 for both comparisons). Pretreatment with ZP followed by progesterone (28 ± 3%) resulted in a significant increase in acrosomal exocytosis versus the control (P < 0.001). Pretreatment with progesterone followed by ZP (36 ± 3%) also resulted in a significant increase in the acrosome reaction versus the control (P < 0.001). However, pretreatment with progesterone followed by ZP resulted in a significant increase in the acrosome reaction when compared with pretreatment with ZP followed by progesterone (P < 0.01). Pretreatment with progesterone followed by ZP increased acrosome reaction significantly when compared with progesterone alone (P < 0.01) and ZP alone (P < 0.01). On the other hand, pretreatment with ZP followed by progesterone did not have a significant effect when compared with progesterone or ZP alone (P > 0.05 for both comparisons). The results of this experiment demonstrated that: (i) progesterone, similar to solubilized ZP, enhances acrosomal exocytosis under microassay conditions; and (ii) sequential treatment with progesterone followed by ZP results in a significantly higher increase in the acrosome reaction than the reverse order, providing evidence for a priming effect (and not a synergistic effect) of progesterone on ZP-induced exocytosis.
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The results of experiment 3a are shown in Figure 3
. The overall results analysed by ANOVA had a significance of P < 0.0001. The calcium ionophore A23187 significantly increased the acrosome reaction from 9 ± 1% (control conditions) to 23 ± 1% (P < 0.001). The solubilized ZP produced an increase in acrosomal exocytosis (47 ± 1%) that was significantly higher than control and calcium ionophore treatment conditions (P < 0.001 versus both conditions). The human follicular fluid-stimulated acrosome reaction (21 ± 1%) was significantly higher than that of the control (P < 0.001), but was not different from that of calcium ionophore (P > 0.5) and was significantly lower than that of ZP (P < 0.001). Pretreatment with follicular fluid followed by ZP (53 ± 1%) resulted in a significantly higher acrosome reaction than the control (P < 0.001), calcium ionophore (P < 0.001), follicular fluid alone (P < 0.001) and ZP alone (P < 0.01). The results of this experiment demonstrated that: (i) follicular fluid, similar to progesterone, is a potent inducer of the acrosome reaction under microassay conditions; and (ii) pretreatment with follicular fluid also results in a priming effect on the solubilized ZP-induced acrosomal exocytosis.
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The results of experiment 3b are shown in Figure 4
. The overall results of the subsequent treatment with follicular fluid and solubilized ZP analysed by repeated measures ANOVA had a significance of P < 0.0002. Upon pretreatment with follicular fluid for 30 min, a time-dependent effect of ZP was observed. The induction of acrosome reaction at 45 and 60 min was significantly higher than that at 15 min (P < 0.01 and P < 0.001 respectively). Induction of the acrosome reaction at 45 and 60 min was also significantly higher than that at 30 min (P < 0.01 and P < 0.001 respectively). There were no significant differences observed for the effect of follicular fluid alone at the different time points. This experiment examined the kinetics of the solubilized ZP-induced acrosome reaction after exposure to human follicular fluid. The results demonstrated that upon exposure to follicular fluid there is a time-dependent effect of solubilized ZP on acrosomal exocytosis, with increased exocytosis after 45–60 min incubation.
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Franken et al. devised a new microassay that is easy and rapid to perform, and facilitates the use of minimal volumes of solubilized ZP (even a single zona) for assessment of the human sperm acrosome reaction (Franken et al., 2000
). The microassay has been validated as compared with the standard macroassay and consequently offers a unique arena to test for the physiological induction of acrosomal exocytosis by the homologous ZP. Moreover, preliminary clinical studies using the microassay have demonstrated that the ZP-induced acrosome reaction (ZIAR) can predict fertilization failure in the IVF setting. The microassay ZIAR can therefore refine the therapeutic approach for male infertility prior to the onset of therapy (Esterhuizen et al., 2001a,b).
We hypothesized that, in human sperm, solubilized ZP triggers acrosomal exocytosis via a transmembrane signalling cascade involving heterotrimeric G proteins (pertussis toxin-sensitive); and that an alternative or complementary pathway may involve regulation of intracellular calcium levels by the modulation of calcium influx. To test this hypothesis, human capacitated sperm were treated with pertussis toxin (which functionally inactivates heterotrimeric Gi protein by ADP-ribosylating its 
-subunit) (Casey and Gilman, 1988) or EGTA (a calcium chelator), prior to induction of the acrosome reaction with solubilized ZP.
Results of experiment 1 confirmed that under the microassay conditions, solubilized ZP induced a high level of acrosomal exocytosis. Importantly, pretreatment with pertussis toxin significantly inhibited the ZP-induced acrosome reaction (although it did not affect basal or spontaneous levels of exocytosis) (Franken et al., 1996). These results confirmed and extended those of Lee et al., Tesarik et al. and Franken et al. who reported on the Gi protein-dependency of the human acrosome reaction triggered by the homologous ZP (Lee et al., 1992; Tesarik et al., 1993; Franken et al., 1996, 2000).
Calcium appears to be essential for several sperm functions. It has been shown that elevated intracellular free calcium concentrations and protein tyrosine phosphorylation are determinants of sperm capacitation and that extracellular calcium modulates tyrosine phosphorylation and tyrosine kinase activity in human sperm (Luconi et al., 1996; Osheroff et al., 1999; Visconti et al., 1999a,b). The acrosome reaction necessary for fertilization in many species also requires an increase in intracellular calcium levels. Incubation of human sperm in a calcium-depleted medium inhibits or delays capacitation, resulting in fewer spontaneous or A23187-induced acrosome-reacted sperm (Perry et al., 1997). In the mouse, ZP3 produces a sustained increase in intracellular calcium leading to the acrosome reaction, probably due to the persistent activation of a calcium influx mechanism during the late stages of ZP3 signal transduction (O'Toole et al., 2000).
In this study, the calcium chelator EGTA significantly inhibited the ZP-induced acrosome reaction. These results demonstrate that extracellular calcium is required for signal transduction resulting in an agonist-induced acrosome reaction. However, the design of these experiments does not allow discrimination between cell surface requirements for calcium (i.e. calcium might be required for sperm–ZP initial attachment, which subsequently leads to induction of exocytosis) or a role of calcium influx itself in the cascade of events leading to the acrosome reaction. Experiments performed in other species have demonstrated the presence and significance of calcium channels and calcium influx in the activation of the acrosome reaction (see above). In addition, two recombinant human ZP3 products have been reported to trigger calcium influx in human sperm (Whitmarsh et al., 1996; Bray et al., 2002). We therefore conclude that our data provide further indirect evidence for the role of calcium influx on the induction of the human acrosome reaction. In our experiments, pretreatment with EGTA did not inhibit the basal or spontaneous acrosome reaction, suggesting that intracellular sources of calcium may be sufficient for sustaining basal levels of exocytosis.
The results of experiments 2 and 3 confirm that human follicular fluid and progesterone (which is present at high concentrations in follicular fluid) are potent stimulators of the acrosome reaction (Blackmore et al., 1990). Controversy surrounds the precise time at which the physiological acrosomal exocytosis occurs, i.e. during exposure to follicular fluid–cumulus cell products or at the level of the ZP. We hypothesized that a population of sperm may undergo exocytosis before reaching the zona, but that the most functional acrosome reaction leading to zona penetration takes place after interaction with ZP3, possibly primed by follicular fluid constituents.
In the mouse, Roldan et al. have elegantly shown that progesterone exerts a priming effect on ZP-induced exocytosis (Roldan et al., 1994). Here, we have provided further data in support of this priming effect as related to progesterone and follicular fluid in human sperm. The progesterone priming effect was evident even after 15 min of preincubation. Moreover, experiments revealed that the priming effect produced by follicular fluid on the ZP-induced acrosome reaction was time-dependent, with maximal results observed in the range of 45–60 min zona incubation following steroid exposure.
In conclusion, our studies have demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by homologous solubilized ZP is dependent on both the activation of Gi protein (pertussis toxin-sensitive) and on the presence of extracellular calcium; and (ii) progesterone and human follicular fluid exert a priming, time-dependent effect on the ZP-induced acrosome reaction. The operative mechanisms downstream of Gi protein activation (ZP-dependent) and to the increase in intracellular calcium levels (ZP and progesterone/follicular fluid-dependent), and cross-talk between such pathways leading to acrosomal exocytosis in human sperm remain to be further characterized.
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4 To whom correspondence should be addressed. E-mail: oehninsc{at}evms.edu
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References |
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Submitted on December 21, 2001; accepted on May 14, 2002.
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