Reagents

Calcium ionophore A23187, GTPgS, GDPbS, lisophosphatidylcholine,  mastoparan, fluorescein isothiocyanate-labelled Pisum sativum lectin (FITC-PSA), were obtained from Sigma Chemical Company (St. Louis, MO). [AlF4]^- was prepared from original stocks.  Streptolysine-O was obtained from Welcome Dagnostics. The culture media was GPM (Gamete Preparation Media, Serono-Spain).Water for all solutions was purified with a batch-fed system (Barnsted EASYpure, Dubuque, IA).

Experimental design

Human semen samples from at least 5 healthy donors of proven fertility (motility >50%, motile spermatozoa  >60x10⁶/ml) were used. After swim-up separation (Makler et al., 1984) for 1 h at 37ºC in an atmosphere of 95% air - 5% CO2 using GPM culture media, highly motile sperm were recovered. The concentration was adjusted to 1-5x106/ml in fresh culture media, and incubation proceeded for additional 2 h at 37ºC and 5% CO2, accounting for a total incubation time of 3 h under capacitating conditions. In different sets of experiments, the following reagents were tested: 1) vehicle (control groups); 2) calcium (0.4-0.7 mM); 3) calcium ionophore A23187 (5 microM); 4) lisophosphatidylcholine (LPC, 25 microM); 5) GTPgS (40 microM); 6) GDPbS (200 microM); 7) [AlF4]^- (5 mM NaF and 100 microM Al2(SO4)3); and 8) mastoparan (MP, 20 microM). Permeabilization was performed at 4ºC after washing the incubated samples with cold PBS for two times, by adding SLO (0.5 UI/ml final concentration). After 15 min the sample was washed again with cold PBS, and finally resuspended in sucrose-buffer with DTT 2.0 mM. Temperature was slowly increased to 37ºC and, after 15 min, all reagents were added to 100 microliters aliquots for 15 additional min. Non permeabilized samples were treated in a similar way, except for the fact they were not permeabilized.

Acrosome reaction assay

The acrosome reaction was evaluated by the FITC-PSA lectin according to Mendoza et al. (1992). Briefly, the cell suspensions were centrifuged for 1 min in a Beckman Microfuge (at least two cycles) and the pellets resuspended in phosphate buffered saline (PBS) for 3 min. A 10 ml drop was then placed on a spotted-slide and air-dried at room temperature. After air-drying, the sperm smears were fixed with cold (4ºC) methanol for 30 sec and incubated in the darkness with 50 mg/ml FITC-PSA in PBS in a moisturized chamber for 30 min at room temperature. After intensive washing with distilled water, unmounted slides were examined on the same day of the staining procedure, and at least 200 live cells were evaluated in a epifluorescence Nikon Optiphot II microscope (100x) according to the following patterns: a) selective staining of the whole acrosome (unreacted cell); b) no staining at all, or staining limited to the equatorial segment (reacted cells).

Statistical analysis

Differences between experimental and control conditions were tested by One-Way ANOVA and Fisher's PSLD tests. Where necessary, percentages were transformed to the arc-sine prior to analysis. Significant differences were those where p was <0.05

Almost all spermatozoa resulted permeabilized since most of them (>99%) were stained by the supravital stain eosin-Y, indicating that the cells were functionally unable of maintaining the stain outside the intracellular space (by definition we could consider the cells functionally dead). We found the non-permeabilized cells to be able of responding to A23187, indicating the acrosomes were functionally competents. Also, when we treated the cells with calcium, we could see that calcium (0.5 mM was significantly different from controls and calcium 0.4 mM, with a plateau at 0.6 mM) increased AR to levels comparable to those reached by intact spermatozoa treated with well known stimulants like LPC and A23187, as can be seen in Figure 1.

Fig. 1: Percentage of acrosome reaction in non-permeabilized and permeabilized spermatozoa. Iono= calcium ionophore A23187; CaCl2= calcium. Different letters indicate significant differences p < .05

Also, G-protein stimulation with mastoparan, GTPgS or AlF increased AR. On the other hand, GDPbS, a G-protein inhibitor, was able of blocking the increase of AR induced by calcium, as can be seen in Figure 2.

Fig. 2: Percentage of acrosome reaction in permeabilized spermatozoa. gamma= GTPgS; AlF= [AlF4]; MP= mastoparan; Ca+beta= calcium + GDPbS. When two reagents are mentioned, the first one was added 15 min before the second one. Different letters indicate significant differences p < .05

Our results suggest that permeabilization of human spermatozoa with SLO does not affect the intrinsic capability of the acrosome to respond to different stimulus, notwithstanding the fact that all spermatozoa could be considered technically dead. In fact, we consider the whole process to be far from physiologically acceptable. The fact is, however, that we do not wanted to obtain a physiologically valid model but one eventually useful to test macromolecules during the cascade of signals conducting to the acrosome reaction. Also, from our data, we reinforce the idea of both a G-protein and calcium participating during the AR.

It is concluded that the model could be eventually useful to study macromolecules involved on the acrosome reaction of human spermatozoa.

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