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Impact of Ca²⁺ Flux Inhibitors on Acrosome Reaction of Hamster Spermatozoa

YI B. HAN

LEI J. ZHENG

From the ^* Center for Human Reproduction, North Shore University Hospital, New York University School of Medicine, Manhasset, New York; Department of Obstetrics and Gynecology, Chinese University of Hong Kong, Hong Kong, China; and Columbia University Center for Women's Reproductive Care, New York, New York.

Correspondence to: Dr Huai L. Feng, Associate Professor, The Center for Human Reproduction, Department of Obstetrics and Gynecology, North Shore University Hospital, 300 Community Dr, Manhasset, NY 11030 (e-mail: hfeng{at}nshs.edu).

Received for publication October 10, 2006; accepted for publication February 26, 2007.

	Abstract

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Ca²⁺ plays a prominent role in the regulation of critical functions of spermatozoa, such as capacitation, acrosome reaction (AR), and fertilization. While there is consensus that Ca²⁺ is essential, researchers have reported conflicting results as to what happens to Ca²⁺ flux across the sperm membrane during capacitation and AR. The purpose of the present study was to further delineate the function of Ca²⁺ channels and their role in sperm capacitation and AR. Epididymides were obtained from healthy adult male hamsters. Spermatozoa were washed with modified Tyrode medium supplemented with 0.3% bovine serum albumin, adjusted to 4.5 x 10⁷ motile sperm/mL and incubated in 200-µL droplets for 4 hours at 37°C in the presence of trifluoperazine (TFP), a calmodulin inhibitor, at 25, 50, 100, or 150 nM; verapamil (VP), a Ca²⁺ channel inhibitor, at 25, 50, 100, or 150 nM; or nifedipine (NF), a voltage-dependent Ca²⁺ channel inhibitor, at 50, 100, 200, or 400 nM. Spermatozoa were assessed for AR by using Coomassie brilliant blue staining techniques. Results indicated that incubation of sperm with Ca²⁺ channel inhibitors for 4 hours significantly reduced AR in the study groups (TFP: 88% ± 2.3%, 65% ± 2.0%, 60% ± 2.2%, 54 % ± 2.2%, respectively; VP: 45% ± 1.3%, 23 % ± 1.2%, 12% ± 1.0%, 8% ± 0.6%, respectively; and NF: 11% ± 0.8%, 9% ± 0.3%, 7.0% ± 0.1%, 6.0% ± 0.1%, respectively) compared with control group (95% ± 3.0%, P < .05). However, increasing the concentrations of TFP and NF did not result in further suppression of AR. In summary, the antagonists of calmodulin and Ca²⁺ channel inhibitors suppress sperm AR.

     Key words: Calcium

	Materials and Methods

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Animals and Specimens

Cauda epididymal spermatozoa were obtained from 15 sexually mature Chinese hamsters. The use of these specimens for the present study was approved by Jilin University School of Animal Science Institutional Review Board.

Sperm Preparation and Incubation

Sperm specimens were collected by perfusion of the cauda epididymis through the distal end of the vas deferens with prewarmed modified Tyrode medium containing 108.76 mM NaCl, 2.70 mM KCl, 0.49 mM MgCl₂, 2.4 mM CaCl₂, 25.07 mM NaHCO₃, 0.32 mM NaH₂PO₄·H₂O, 5.56 mM glucose, 1.0 mM sodium pyruvate, 10.0 mM sodium lactate, and 4 mg/mL bovine serum albumin (BSA; pH 7.5). After centrifugation at 200 x g for 5 minutes at room temperature, the supernatant was removed; pelleted cells were resuspended in fresh medium. Sperm concentration was determined using a hemocytometer to adjust to 4 x 10⁵ sperm/mL. In the control group, the suspension specimen was incubated in 200-µL droplets (modified Tyrode medium and 4 mg/mL BSA) under paraffin oil for 4 hours at 37°C in a moist atmosphere of 5% CO₂. These conditions are known to induce capacitation and AR (Ain et al, 1999). In the experimental groups, according to prior studies (Fraser and McIntyre, 1989; Li and Chen, 1996), the suspension specimen was incubated in 200-µl droplets containing trifluoperazine (TFP), a calmodulin inhibitor, at 25, 50, 100, or 150 nM; verapamil (VP), a Ca²⁺ channel inhibitor, at 25, 50, 100, or 150 nM; or nifedipine (NF), a voltage-dependent Ca²⁺ channel inhibitor at 25, 50, 100, 200, or 400 nM for up to 4 hours at 37°C in a moist atmosphere of 5% CO₂. Aliquots of sperm were removed from each group for assessment of sperm acrosomal status by using Coomassie brilliant blue staining techniques. All tests were repeated at least 3 times.

Acrosome Staining Using Coomassie Brilliant Blue

Sperm was air-dried onto glass slides, fixed with 5% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 15 minutes, and washed once with PBS. Slides were stained for 5 minutes with aqueous 0.25% Coomassie brilliant blue R-250 (Amresco Inc, Solon, Ohio) in 10% glacial acetic acid and 25% methanol; they were then rinsed with H₂O and covered with coverslips under mounting media (Lerner Labs Inc, Pittsburgh, Pa). This method stains the acrosomal cap blue in acrosome-intact sperm but does not stain the acrosome region in acrosome-reacted sperm (Feng et al, 1997). Each acrosome assessment represents 5 to 6 microscopic fields with 80 to 100 sperm in each field.

Data Analysis

Results were analyzed using 1-way analysis of variance and ² and Fisher's exact tests when applicable. P < .05 was accepted as significant.

	Results

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Incubation of sperm with each antagonist for 4 hours significantly reduced AR in the experimental groups. In the TFP group, AR rates were 88% ± 2.3%, 65% ± 2.0%, 60% ± 2.2%, and 54 % ± 2.2%, respectively (Figure 1A); in the VP group, they were 45% ± 1.3%, 23% ± 1.2%, 12% ± 1.0%, and 8% ± 0.6%, respectively (Figure 1B); and in the NF group, they were 12% ± 0.9%, 11 % ± 0.8%, 9% ± 0.3%, 7.0% ± 0.1%, and 6.0 ± 0.1%, respectively, compared with the control group (95% ± 3.0%) (Figure 1C). However, dose response could not be demonstrated. Increasing the concentration of each of the inhibitors (TFP or NF) did not result in further AR suppression.

View larger version (14K): [in this window] [in a new window]   Figure 1. Effects on the acrosomal status of sperm incubated with (A) trifluoperazine, (B) verapamil, or (C) nifedipine for 4 hours in vitro. The data represent the percentage of acrosome-reacted sperm cells as determined by Coomassie brilliant blue staining and are the means (± SD) of 3 separate trials, which counted 200 cells per treatment per trial. Values with asterisks differ significantly from values in the control group. *P < .05, **P < .01 using the chi-square test.

	Discussion

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Calcium deposits have been shown to be more abundant in the plasma membrane, acrosome membrane, AR vesicles, and matrices of the acrosome and mitochondria in capacitated sperm (Breitbart et al, 1984; Watson et al, 1995; Feng et al, 2006). Studies on Ca²⁺ current indicate that internalization of Ca²⁺ leading to a rise in [Ca²⁺]_i is required during both capacitation and acrosomal exocytosis in mammalian spermatozoa (Fraser and McDermott, 1992). Although several mechanisms with the potential to modulate [Ca²⁺]_i have been reported (Fraser, 1995a), their precise role in sperm physiology remains controversial. Ca²⁺-ATPase has been located in the plasma membrane, outer and inner membranes of the acrosome, and outer mitochondria membrane in hamster spermatozoa (Feng et al, 2006). However, Ca²⁺-ATPase was only weakly reacted in the plasma membrane of sperm heads and outer mitochondria membranes of the midpiece in capacitated hamster spermatozoa (Feng et al, 2006). These observations provide direct evidence that decreases in Ca²⁺-ATPase activity occurs during hamster sperm capacitation, enabling a rise in [Ca²⁺]_i to the threshold required for capacitation, which supports previous studies showing that Ca²⁺-ATPase antagonists or inhibitors accelerated the onset of capacitation and AR in human (DasGupta et al, 1994; Perry et al; 1997; Rossato et al, 2001), mouse (Fraser and McDermott, 1992; Susan et al, 1996) and guinea pig (Roldan and Fleming, 1989; Li and Chen, 1996) spermatozoa. A prior study indicated that sperm preincubated with TFP, a calmodulin antagonist, were significantly more fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitation in mouse sperm (Adeoya-Osiguwa and Fraser, 1996). However, our study suggested that TFP does not stimulate hamster sperm capacitation by blocking calmodulin's function, thus decreasing Ca²⁺-ATPase activity; instead, it was shown that TFP inhibits sperm AR, suggesting that calmodulin may not have a direct impact on Ca²⁺-ATPase activity in hamster sperm, much like guinea pig sperm (Li and Chen, 1996). Also, TFP may directly interfere with Ca²⁺-ATPase or Ca²⁺ channel activity, resulting in the blockage of sperm AR. Furthermore, our data indicated that VP, an inhibitor of plasma Ca²⁺ channels, and NF, an inhibitor of voltage-dependent Ca²⁺ channels, significantly reduce sperm AR in comparison with TFP; one suggested mechanism is the cessation of Ca²⁺ influx via blockage of plasma Ca²⁺ channels by VP and NF, thus resulting in diminished [Ca²⁺]_i and consequently decreasing AR. These conclusions are supported by previous studies in human (Brandelli et al, 1996; Kirkman-Brown et al, 2003) and mouse (Feng et al, 1998) spermatozoa.

In summary, the antagonists of calmodulin and Ca²⁺ channel inhibitors suppress sperm AR, which may affect the function of Ca²⁺-ATPase and Ca²⁺ channels, enabling a rise in [Ca²⁺]_i to a threshold required for capacitation and acrosomal exocytosis.

	Footnotes

 
This study was presented at the 62nd Annual American Society for Reproductive Medicine meeting in New Orleans, La, October 21–25, 2006.

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