Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

In these investigations, unfertilized oocytes used were donated by patients from Assisted Reproduction Technology Centre, Army Hospital Research & Referral, New Delhi following project approval by the respective Institutional Human Ethical Committees and signed patient consent. The follicular fluid from women undergoing In Vitro Fertilization (IVF) treatment was aspirated under general anaesthesia and aseptic conditions. Oocyte-cumulus complex (OCC) were immediately separated under stereo zoom microscope (Zeiss, Baden-Wuerttenberg, Germany) and maintained in Universal IVF Medium (MediCult a/s, Mellehaven 12, Denmark) under liquid paraffin (MediCult a/s) and were inseminated with 0.1 × 10⁶ motile sperm per OCC. Fertilization was confirmed after 17-24 hr by appearance of two pronuclei or second polar body. Those oocytes that failed to show the two pronuclei or the second polar body were further incubated for 12 hr and in absence of evidence of fertilization, they were stored in Embryo Freezing Medium (MediCult a/s) in liquid nitrogen until used in the present study. Prior to use, the oocytes were thawed, washed three times in 50 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS) and vigorously pipetted with small bore glass pipette to remove ZP from oocyte. The suspension was centrifuged at 1800 ×g for 15 minutes to pellet down ZP. The zonae were re-suspended in PBS and heat-solubilized at 70°C for 90 min. This preparation was designated as human SIZP.

All experiments using human spermatozoa were carried out under informed consent and following the clearance from the Institutional Bio-safety and Human Ethical Committee. Semen samples were collected from healthy donors after 3 days of sexual abstinence. Semen samples were assessed for volume, total sperm count, sperm morphology and sperm motility as per the WHO guidelines [18]. Semen samples showing sperm count of less than 20 million/ml or sperm motility less than 70% were not included in the present study. Semen samples from individual donors were processed separately and subjected to liquefaction at room temperature (RT) for 30 min. The motile sperm were isolated by two-step Percoll density gradient as described previously [19]. The sperm (10 × 10⁶ cells/ml) were capacitated in Biggers-Whitten-Whittingham medium [20] supplemented with 2.6% BSA for 6 h at 37°C with 5% CO₂ in humidified air in aliquots of 1 ml. Capacitated sperm (1 × 10⁶ in BWW + 0.3% BSA) were incubated at 37°C with 5% CO₂ in humidified air for 1 hr in presence of SIZP in a total reaction volume of 100 μl. For measurement of spontaneous induction of acrosome reaction, sperm were also incubated with BWW + 0.3% BSA alone. Calcium ionophore (10 μM, A23187; Sigma-Aldrich Inc., St. Louis, MO, USA) served as a positive control in all the experiments. Post-incubation, the sperm were washed with 50 mM PBS pH 7.4, assessed for sperm viability by one step eosin-nigrosin staining method [21] and 20 μl aliquots were spotted on poly-L-Lysine coated slides (Sigma-Aldrich Inc.) in duplicates. The spots were air-dried, fixed in chilled methanol for 30 seconds and stained with 5 μg/ml tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA; Vector Laboratories Inc., Burlingame, CA, USA) for 30 min at RT. Any spermatozoa that demonstrated complete loss of TRITC-PSA staining in the acrosome or revealed staining at the equatorial region was classified as acrosome-reacted. Sperm showing TRITC fluorescence in the acrosomal region of the head were classified as acrosome intact. All the slides were read "blind" with coded samples under Nikon Eclipse 80 i epifluorescence microscope (Nikon, Chiyoda-ku, Tokyo, Japan) using an oil immersion objective. Two hundred sperm were scored for every spot and the percentage of acrosome reaction was calculated by dividing the number of acrosome-reacted sperm by the total number of sperm counted and multiplied by hundred. Induction of acrosome reaction at an optimized dose of SIZP was evaluated using semen samples from six different donors.

Changes in [Ca²⁺]_i were analyzed with the fluorescent probe Fluo-3 acetoxymethyl (AM) ester (Molecular Probes, Eugene, OR, USA). Capacitated sperm (10 × 10⁶) were loaded with 2 μM fluo-3 AM containing 1 μM pluronic acid F-127 (Molecular Probes; for proper dispersal of dye) for 1 hr at 37°C with 5% CO₂ in air. Labelled sperm were then kept for half an hour at 37°C with 5% CO₂ in air for de-esterification of dye. Labelling and de-esterification of Fluo-3 AM in capacitated sperm was performed in BWW medium whereas assay was performed in BWW medium supplemented with 0.3% BSA. Capacitated sperm (1 × 10⁶/well) were added in 96 well black plates (BMG Technologies, Offenburg, Germany). The baseline fluorescence measurements were performed at an excitation wavelength of 480 nm and an emission of 520 nm for ~200 sec followed by addition of SIZP and continued fluorescence measurements for next ~10 minutes. The [Ca²⁺]_i was calculated by using the Grynkiewicz equation [Ca²⁺]_i = K_d (F-F_min)/(F_max-F), where K_d is the dissociation constant of the Ca²⁺-fluo-3 complex (390 nM), and F represents the fluorescence intensity of the cells [22]. F_max represents the maximum fluorescence (obtained by treating cells with 1% Triton-X) and F_min corresponds to the minimum fluorescence (obtained in the presence of 8 mM EGTA). Ca²⁺ levels [nM] have been presented as the change in intracellular calcium, Δ [Ca²⁺]_i by calculating difference between peak [Ca²⁺]_i and resting [Ca²⁺]_i before stimulation. Resting [Ca²⁺]_i represent the average of sperm [Ca²⁺]_i for 200 sec preceding SIZP addition. All measurements were carried out in a Fluostar Optima Spectrofluorimeter (BMG Technologies).

To delineate the involvement of different type of Voltage Operated Calcium Channels (VOCCs) during SIZP mediated induction of acrosome reaction, 1 × 10⁶ capacitated sperm were pre-treated with Pimozide (10 and 20 μM) or Mibefradil (5 and 10 μM) as T-Type Ca²⁺ channel blocker (CCB); Verapamil (10 and 20 μM) or Nifedipine (10 and 20 μM) as L-Type CCB; for 10 min at 37°C with 5% CO₂ in humidified air prior to the addition of SIZP. The concentrations of the various inhibitors employed in these experiments were based on previously published studies [6, 23‐26] and inhibitors were procured from Sigma-Aldrich Inc. In addition, effect of prior treatment of capacitated human sperm with Pimozide and Verapamil on the levels of [Ca²⁺]_i in response to SIZP were also determined by fluorimetric assay as described above.

To understand the mechanism of action of human SIZP mediated acrosome reaction, 1 × 10⁶ capacitated spermatozoa were pre-treated with various pharmacological inhibitors such as Picrotoxin (50 and 100 μM) - GABA_A receptor antagonist [27]; Pertussis toxin (PTX, 0.1 μg/ml) - G_i protein-coupled receptor pathway inhibitor [6, 16, 28, 29]; Herbimycin-A (10 and 15 μM) - tyrosine kinase inhibitor [6, 30]; Chelerythrine chloride (2 and 3 μM) - protein kinase C inhibitor [6, 30]; Wortmannin-A (50 and 100 nM) - phosphoinositide 3-kinase (PI-3 kinase) inhibitor [31]; H-89 (20 μM) - cAMP dependent protein kinase A inhibitor [6, 30] for 1 hr (except Picrotoxin, pre-treated for 10 min) at 37°C with 5% CO₂ in humidified air prior to the addition of human SIZP. To study the relevance of extra-cellular Ca²⁺, capacitated sperm were either pre-incubated for 10 min with 8 mM EGTA or added at the same time as SIZP. All the above inhibitors were procured from Sigma-Aldrich Inc.

The results pertaining to SIZP mediated induction of acrosome reaction are presented as mean ± SEM and statistical analysis was done by comparing the means of the medium control (BWW + 0.3% BSA)/vehicle control and experimental sets or within two experimental groups by using paired Student's t-test/Wilcoxon signed rank test. A value of p < 0.05 was considered to be statistically significant.

A significant increase in the induction of acrosomal exocytosis of capacitated human sperm was observed with a concomitant increase in number of zonae equivalent (SIZP) used per reaction, as compared to PBS (solvent used in preparation of SIZP; Table 1). As low as ~1 zona equivalent was able to induce statistical significant induction of acrosome reaction in capacitated human sperm. However, no further increase in acrosomal exocytosis was observed with SIZP preparation from more than ~5 zonae per reaction. Subsequently, 5 zonae equivalent SIZP was used in all experiments. Capacitated sperm prepared from 6 different donors on incubation with optimized concentration of human SIZP showed a significant (p = 0.007) increase in induction of acrosome reaction (Table 1).

An increase in [Ca²⁺]_i, after coming in contact with ZP, is a prerequisite for induction of acrosomal exocytosis in mammalian sperm [32]. In the present study, SIZP was also able to elicit an increase in [Ca²⁺]_i after incubating with fluo-3/AM labelled capacitated human sperm (Fig. 1). To decipher the type of VOCCs playing an important role in human SIZP mediated increase in initial [Ca²⁺]_i surge as well as subsequent induction of acrosome reaction, pharmacological inhibitors for L- and T- type VOCCs were employed. Prior-incubation (10 min) of fluo-3/AM labelled capacitated human sperm with Pimozide (20 μM; T- type VOCC inhibitor) inhibited the SIZP mediated initial increase in [Ca²⁺]_isurge, whereas Verapamil (10 μM; L- type VOCC inhibitor) failed to do so (Fig. 1).

To further assess the importance of these VOCCs, induction of acrosome reaction in capacitated human sperm was quantitated after incubation with SIZP in presence or absence of pharmacological inhibitors of L- or T-type specific VOCC. Pre-incubation of capacitated human sperm with T-type VOCC inhibitors, Pimozide (10 and 20 μM) or Mibefradil (5 and 10 μM) significantly reduced the SIZP mediated induction of acrosome reaction whereas L-type VOCC inhibitors, Nifedipine (10 and 20 μM) or Verapamil (10 and 20 μM) failed to inhibit the same (Fig. 2a, b).

Pre-incubation of capacitated human spermatozoa with EGTA (8 mM) for 10 min was able to significantly (p = 0.001) inhibit SIZP mediated induction of acrosome reaction (Table 2). Further, capacitated sperm after re-suspension in the EGTA medium were immediately exposed to human SIZP. Under these experimental conditions, 16 ± 0.6% sperm showed acrosome reaction in presence of EGTA as compared to 36.1 ± 1.0% in its absence (p = 0.0001), suggesting the role of extra-cellular calcium in SIZP mediated induction of acrosomal exocytosis in human sperm. Addition of 8 mM EGTA led to negligible levels of free calcium in the reaction medium as analyzed by Maxchelator programme [33].

SIZP mediated induction of acrosomal exocytosis (31.1 ± 1.2%) was inhibited in presence of Pertussis toxin (0.1 μg/ml) to a statistically significant extent (21.1 ± 0.8%; p = 0.02; Table 2). Acrosomal exocytosis mediated by SIZP was also inhibited by prior incubation of capacitated human sperm with either 50 or 100 μM GABA_A receptor antagonist, Picrotoxin (from 28.9 ± 1.0% to 23.4 ± 0.5% & 21.3 ± 0.5% respectively) and cAMP dependent protein kinase A inhibitor, H-89 (from 31.1 ± 1.2% to 19.8 ± 0.9%) (Table 2). In addition, pre-incubation of capacitated human sperm with inhibitors of other kinases such as, PKC (Chelerythrine chloride), PI3-kinase (Wortmannin A) and tyrosine kinase (Herbimycin A) also led to inhibition of SIZP mediated acrosomal exocytosis (Table 2).