Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Abstract

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.

Figure 1. Assessment of the participation of nitrergic system on sperm release from bovine oviductal monolayers (BOEC).

Sperm cells and BOEC were co-cultured and then incubated for 15 min with BSA-free sp-TALP alone (control), increasing concentrations of A) NOC-18 (a NO donor) or B) L-Arginine (10 mM; NO synthase substrate). Bars indicate the number of spermatozoa that remained attached to the monolayers and represent the mean ± S.E.M. of bound spermatozoa/0.11 mm² monolayer (n = 6), a≠b p<0.05.

Figure 2. Assessment of participation of the nitrergic system on sperm release induced by anandamide.

Sperm cells and BOEC were co-cultured and then incubated for 15 min with BSA-free sp-TALP alone (control), AEA (1 nM) or MetAEA (1.4 nM) and L-NAME (1 µM; NO synthase inhibitor) or Hemoglobin (Hb) (30 µg/ml; NO scavenger). Bars indicate the number of spermatozoa that remained attached to the monolayers and represent the mean ± S.E.M. of bound spermatozoa/0.11 mm² monolayer (n = 6), a≠b p<0.05.

Figure 3. Effect of L-NAME or Hemoglobin on bull sperm capacitation induced by AEA.

Spermatozoa were incubated for 45 min at 38.5°C in 0.3% BSA sp-TALP medium with AEA (1 nM) and L-NAME (1 µM) or Hb (30 µg/ml). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). A: Assessment of sperm capacitation by CTC; T0 and T45: 0.3% BSA sp-TALP at 0 and 45 min (control) incubation respectively (n = 6). B: Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. T45: 0.3% BSA sp-TALP (control). After capacitation, spermatozoa were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 6). Data are expressed as mean ± SEM. a≠b, p<0.05.

Figure 4. Effect of L-NAME or Hemoglobin on bull sperm capacitation induced by MetAEA.

Spermatozoa were incubated for 45 min at 38.5°C in 0.3% BSA sp-TALP medium with MetAEA (1.4 nM) and L-NAME (1 µM) or Hb (30 µg/ml). Bars indicate the percentage of capacitated spermatozoa (A: % pattern B of CTC; B: % acrosome reacted spermatozoa). A: Assessment of sperm capacitation by CTC; T0 and T45: 0.3% BSA sp-TALP at 0 and 45 min (control) incubation respectively (n = 6). B: Assessment of sperm capacitation by LPC-induced acrosome reaction (AR)-PSA-FITC. T45: 0.3% BSA sp-TALP (control). After capacitation, spermatozoa were incubated for 15 min either with or without LPC to induce AR. Bars show percentage of spermatozoa that underwent LPC-induced AR minus the percentage of spermatozoa that underwent spontaneous AR (n = 6). Data are expressed as mean ± SEM. a≠b, p<0.05.

Figure 5. Determination of NO levels in bull spermatozoa.

Sperm samples were incubated for 60 min at 38.5°C in 0.3% BSA sp-TALP containing 0.1 mM L-Arginine and 5 µM of DAF-FM diacetate and untreated (control) or treated with MetAEA (1.4 nM), MetAEA+L-NAME (1 µM). Spermatozoa were fixed and the fluorescent complex was measured by flow cytometry. A) A representative photograph showing fluorescence in spermatozoa, indicating the content of intracellular NO (Magnification, ×600); B) Fluorescence data are expressed as mean fluorescence (percentage of control at 45 min incubation, control adjusted to 100%). Data are expressed as mean ± SEM (n = 8). a≠b, p<0.05.

Figure 6. Participation of CB1 and TRPV1 in NO production during bull sperm capacitation by AEA.

Sperm samples were incubated for 60 min at 38.5°C in 0.3% BSA sp-TALP containing 0.1 mM L-Arginine and 5 µM of DAF-FM diacetate and untreated (control) or treated with MetAEA (1.4 nM) and/or SR141716A (SR1: CB1 antagonist (0.1 nM)) or Capsazepine (CZP: TRPV1 antagonist (10 nM)). Spermatozoa were fixed and the fluorescent complex was measured by flow cytometry. Fluorescence data are expressed as mean fluorescence (percentage of control at 45 min incubation, control adjusted to 100%). Data are expressed as mean ± SEM (n = 5). a≠b, p<0.05.

Figure 7. Localization of nNOS and eNOS in bovine spermatozoa.

Spermatozoa were incubated with the primary antibodies against nNOS (panel A) or eNOS (panel C); B and D) phase contrast. Controls were performed incubating sperm cells with IgG fractions from non-immunized rabbits at the same concentration that the primary antibody (data not shown) (n = 3); Scale bar: 10 µm (Magnification, ×600). Arrows indicate the immunoreactive staining of the NOS antibodies.