Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

Progesterone, bovine serum albumin (BSA), RPMI-1640 medium, dimethyl BAPTA, RU-38486 and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) were from Sigma (Madrid, Spain). Fura-2 acetoxymethyl ester (fura-2/AM) and thapsigargin were from Invitrogen (Barcelona, Spain). Cytochalasin D and jasplakinolide were from Calbiochem (Darmstadt, Germany). Anti-progesterone receptor c262 mouse monoclonal antibody (PR c262) was obtained from Santa Cruz (Santa Cruz Biotechnology, Germany). All others reagents were of analytical grade.

Human semen was obtained from 37 healthy volunteers and 33 asthenozoospermic men at the Extremadura Center of Human Assisted Reproduction (Badajoz, Spain), as approved by local committees and in accordance with the Declaration of Helsinki. This study was approved by the institutional review board of the University of Extremadura and by the ethics committee of the Infantile Hospital (Badajoz, Spain). Each subject was ascertained to be in good health by means of their medical history and a clinical examination including routine laboratory tests and screening. The subjects all were nonsmokers, were not using any medication, and abstained from alcohol. Informed consent was obtained from all patients. Samples were collected by masturbation after 4 or 5 days of sexual abstinence and were allowed to liquefy at 37°C for 30 minutes. Semen was washed twice in RPMI medium (250 × g, 10 min), the supernatant was discarded, and the sperm pellet was resuspended in Na-HEPES solution containing the following (in mM): NaCl, 140; KCl, 4.7; CaCl₂, 1.2; MgCl₂, 1.1; glucose, 10; and HEPES, 10 (pH 7.4). The classical semen parameters of spermatozoa concentration, motility, and morphology were examined according to World Health Organization criteria [26]. Sperm concentration and motility were assessed by a computer assisted semen analysis (CASA) system. Our CASA system was based upon analysis of 25 consecutive, digitalized photographic images obtained from a single field at a 200 × magnification on dark field. The percentages of progressive motility were measured. The main criterion for classification of asthenozoospermic men was low sperm motility [27]. Normozoospermia was indicated by a sperm concentration of ≥ 20 × 10⁶ cells/mL (mean ± SD = 62 ± 30 × 10⁶ cells/mL), a progressive motility (grade a + b sperm motility) ≥ 50% (mean ± SD = 54.2 ± 4.1%) and a normal sperm morphology ≥ 14% (mean ± SD = 17 ± 3.6%). Asthenozoospermia was characterised by a sperm concentration of ≥ 20 × 10⁶ cells/mL (mean ± SD = 42 ± 16 × 10⁶ cells/mL) and a reduced forward motility (grade a+b sperm motility) <50% (mean ± SD = 23.3 ± 12.2%) or absent sperm motility, irrespective of the morphology results.

Cells were loaded with fura-2 by incubation with 4 μM fura-2 acetoxymethyl ester (Fura-2 AM) for 30 minutes at room temperature, according to a procedure published elsewhere [28]. Once loaded, cells were washed and used within the next 2–4 hours. Fluorescence was recorded from 2 mL aliquots of magnetically stirred cellular suspension (2 × 10⁸ cells/mL) at 37°C by using a Shimadzu spectrofluorophotometer with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca²⁺]_c were monitored by using the fura-2 340/380 nm fluorescence ratio and were calibrated according to the method of Grynkiewicz et al. [29]. In experiments where calcium-free medium is indicated, calcium was omitted and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) was added.

Calcium entry and release were estimated using the integral of the rise in [Ca²⁺]_c for 2.5 min after addition of CaCl₂ or progesterone + thapsigargin, respectively [30]. Both calcium entry and release are expressed as nanomolar taking a sample every second (nM·s), as previously described [31].

In the absence of extracellular calcium (calcium-free medium), fura-2-loaded human spermatozoa were treated with 20 μM progesterone plus 1 μM thapsigargin. In spite of the fact that the presence of sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) in sperm is still debated, we have used thapsigargin, a well-known SERCA inhibitor, to be sure that intracellular calcium stores were not refilled. In this regard, we have previously reported that human platelets possess two separate agonist-releasable calcium stores differentiated by the distinct sensitivity to thapsigargin [32, 33]. As shown in Figure 1A, treatment with progesterone and thapsigargin induced a typical transient increase in [Ca²⁺]_c due to calcium release from internal stores in spermatozoa from normospermic men. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal was much smaller compared to calcium signal obtained in spermatozoa from normospermic men (Figure 1B). Similar results were obtained when spermatozoa were treated with progesterone alone (insets Fig. 1A and 1B) The integral of the rise in [Ca²⁺]_c above basal for 2.5 min after addition of progesterone plus thapsigargin taking data every second were 13072.7 ± 697.1 and 5926.5 ± 475.3 nM·s in normospermic and asthenozoospermic men, respectively (Figure 1C; n = 7; P < 0.05). In addition, spermatozoa were loaded with dimethyl BAPTA, an intracellular calcium chelator, by incubating the cells for 30 minutes at 37°C with 10 μM dimethyl BAPTA-AM. As expected, dimethyl BAPTA loading prevented progesterone-evoked [Ca²⁺]_c elevations in both normospermic (Figure 1A) and asthenozoospermic (Figure 1B) men.

Moreover, we evaluated the effect of 20 μM progesterone on progressive sperm motility measured by CASA system after 30 min of incubation. The treatment with progesterone caused a significant increase in the percentage of progressive motility in human spermatozoa from normospermic patients (54.2 ± 4.1 and 70.5 ± 2.3% in untreated and progesterone-treated spermatozoa, respectively), whereas progesterone was unable to modify the motility in spermatozoa from asthenozoospermic patients (23.3 ± 12.2 and 27.5 ± 10.3% in untreated and progesterone-treated spermatozoa, respectively).

Figure 2A demonstrates that the increase of [Ca²⁺]_c induced by progesterone plus thapsigargin was also observed in the presence of extracellular calcium ([Ca²⁺]₀ = 1.2 mM). In addition, we tested if progesterone receptor antibodies or antagonists would reverse the stimulatory effects of progesterone on calcium signal in normospermic spermatozoa. Preincubation of fura-2 loaded spermatozoa from normospermic patients with both the anti-progesterone receptor c262 antibody (PR c262) (1:10, final concentration 100 μg/ml) and the progesterone receptor antagonist RU-38486 (50 μM) for 30 min significantly reduced the progesterone-induced calcium release (Figure 2B). This clearly demonstrates that the blockade of progesterone receptors reduces the calcium mobilization induced by progesterone, and therefore normospermic spermatozoa behave as asthenozoospermic-like spermatozoa.

Interestingly, subsequent addition of calcium (300 μM) to the suspension of progesterone plus thapsigargin-treated spermatozoa resulted in a detectable increase in [Ca²⁺]_c indicative of calcium entry (Figure 3A). Similarly, subsequent calcium entry was significantly reduced (P < 0.05) in comparison to normospermic patients (Figure 3A). The integral of the rise in [Ca²⁺]_c above basal for 2.5 min after addition of calcium taking data every second were 52003.2 ± 3219.4 and 17770.3 ± 2084.1 nM·s in normospermic and asthenozoospermic patients, respectively (Figure 3B; n = 7; P < 0.05).

Cytochalasin D, a widely utilized membrane-permeant inhibitor of actin polymerization which binds to the barbed end of actin filaments [34], and jasplakinolide, a cell-permeant peptide isolated from Jaspis johnstoni which induces polymerization and stabilization of actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses [35, 36], are useful tools to further study the role of the actin cytoskeleton in store-mediated calcium entry. As shown in Figure 4A, pretreatment of human spermatozoa with both 10 μM cytochalasin D for 40 min and 10 μM jasplakinolide for 30 min at room temperature significantly diminished (p < 0.05) calcium entry evoked by depletion of internal calcium stores induced by progesterone plus thapsigargin in normospermic patients. The integral of the rise in [Ca²⁺]_c above basal for 2.5 min after addition of calcium taking data every second were 28842.4 ± 2519.3 and 36256.1 ± 3129.7 nM·s in spermatozoa treated with cytochalasin D or jasplakinolide, respectively (Figure 4B; n = 7; P < 0.05).

However, these treatments proved to be ineffective at modifying calcium entry in patients with asthenozoospermia (Figure 5A). The integral of the rise in [Ca²⁺]_c above basal for 2.5 min after addition of calcium taking data every second were 20556.1 ± 2521.6 and 17175.3 ± 1624.9 nM·s in spermatozoa treated with cytochalasin D or jasplakinolide, respectively (Figure 5B; n = 7), which closely suggest that cytochalasin D and jasplakinolide are unable to affect the calcium entry evoked by depletion of intracellular calcium pools induced by progesterone plus thapsigargin in asthenozoospermic spermatozoa.