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First published online 6 September 2005
doi: 10.1242/jcs.02536

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Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction

¹ Department of Biomedical Sciences, University of Teramo, Pizza A. Moro 45, 64100 Teramo, Italy
² Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy

View larger version (18K): [in a new window]   Fig. 1. Cannabinoid and vanilloid receptors in sperm cells. (A) Saturation curves of [3H]CP55.940 binding to mouse brain, mouse spleen or boar sperm membranes. (B) Effect of CB1 and CB2 receptor antagonists SR141716 and SR144528, and of natural (AEA) or synthetic (Met-AEA) agonists on the binding of 400 pM [3H]CP55.940 (100% as in A). *P<0.01 versus control (CTR). (C) Saturation curves of [3H]RTX binding to boar sperm membranes, alone or in the presence of 1 µM CPZ. The effect of 1 µM AEA (#) or 1 µM Met-AEA (@) on the binding of 1600 pM [3H]RTX is also shown. In all panels, vertical bars represent the s.d.

View larger version (26K): [in a new window]   Fig. 2. Expression of CB1, CB2 and TRPV1 receptors, and FAAH, in sperm cells. (A) Western blot analysis of sperm cell extracts (20 µg/lane) reacted with specific polyclonal antibodies. Gels were overstained in order to check for antibody specificity. Molecular mass markers are shown on the right side. (B) ELISA test of resting or capacitated sperm cells, reacted with the same antibodies as in A. Values were expressed as percentage of the resting cells, set to 100 (100%=0.340±0.040, 0.270±0.030, or 0.300±0.030 absorbance units at 405 nm for CB1R, TRPV1 or FAAH, respectively). In B, horizontal bars represent the s.d.; *P<0.05, **P<0.01 versus resting sperm cells.

View larger version (15K): [in a new window]   Fig. 3. AMT and FAAH activity in sperm cells. (A) Dependence of AMT activity on AEA concentration, alone or in the presence of the AMT inhibitor VDM11 at 10 µM. (B) Dependence of FAAH activity on AEA concentration, alone or in the presence of the FAAH inhibitor MAFP at 100 nM. In both panels, vertical bars represent the s.d.

View larger version (46K): [in a new window]   Fig. 4. Immunocytochemical analysis of CB1R receptors (A), TRPV1 receptors (B) and FAAH (C), in ejaculated boar sperm cells. Both receptors and the AEA hydrolase FAAH are localized on the post acrosomal region of the sperm head and on the whole middle region. Scale bar: 1 µm.

View larger version (16K): [in a new window]   Fig. 5. Time-dependent changes of capacitation, assessed by chlortetracycline (CTC) staining, in spermatozoa incubated for 6 hours in the absence (CTR) or presence of 1 µM Met-AEA, 0.1 µM SR141716 (the CB1R antagonist) or both. Standard deviation values range amongst experiments from ±2.0 to ±3.1. *P<0.01 versus CTR at time 0; #P<0.01 versus CTR at the corresponding time point.

View larger version (34K): [in a new window]   Fig. 6. Effect of Met-AEA on spontaneous and zona pellucida (ZP)-induced acrosome reaction (AR) detected by FITC-PSA staining. (A) Percentage of AR in spermatozoa capacitated in vitro for 4 hours with 1 µM Met-AEA, 0.1 µM SR141716 or both. (B) Spontaneous and ZP-induced AR recorded in spermatozoa incubated in the absence or presence of 1 µM Met-AEA, 1 mM 8Br-cAMP or both. In A and B the maximum standard deviation was ±1.5 and ±2.0, respectively; *P<0.01 versus CTR, **P<0.01 versus Met-AEA for ZP-induced AR. Samples were not statistically different (P>0.05) for spontaneous AR.

View larger version (40K): [in a new window]   Fig. 7. Role of AMT and TRPV1 in acrosome reaction (AR). Spontaneous or ZP-induced AR recorded in spermatozoa capacitated in vitro for 4 hours in the absence (CTR) or presence of 1 µM Met-AEA, 1 µM CPZ, 10 µM VDM11 or their combinations. Standard deviation values ranged amongst experiments from 0.8 to 3.6. Statistical analysis of spontaneous AR data revealed the following: P<0.01 for CTR versus CPZ, CPZ versus CPZ plus VDM11, and CPZ versus CPZ plus VDM11 plus Met-AEA; P<0.05 for CTR versus Met-AEA plus CPZ, and CPZ versus Met-AEA plus CPZ. Statistical analysis of ZP-induced AR revealed the following: P<0.01 for CTR versus Met-AEA, CTR versus CPZ, CTR versus Met-AEA plus CPZ, CTR versus Met-AEA plus VDM11, and CTR versus Met-AEA plus CPZ plus VDM11.