Anandamide Induces Sperm Release from Oviductal Epithelia through Nitric Oxide Pathway in Bovines
Claudia Osycka‐SalutMaría Gracia GervasiElba N. PereyraMaximiliano CellaMaria L.C. RibeiroA.M. FranchiSilvina Pérez‐Martínez
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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.Keywords:
Capacitation
Anandamide
Oviduct
Acrosome reaction
A complex process of maturation called capacitation is an essential step for spermatozoa to fertilize oocytes. Recent studies have shown that reactive oxygen species (ROS) can enhance the capacitation of human spermatozoa and sperm-zona interaction. We have investigated whether hydrogen peroxide (H2O2) could trigger capacitation of human spermatozoa and the acrosome reaction. The addition of catalase, a specific H2O2 scavenger, at the beginning of the capacitation process decreased the levels of both hyperactivation and induced-acrosome reaction whereas catalase added 15 min before the induction of the acrosome reaction by the calcium ionophore had no effect. Supplementation of the medium with H2O2 resulted in increased levels of hyperactivation and the acrosome reaction, whereas H2O2 added 15 min before induction of the acrosome reaction did not have any stimulatory effect. These results suggest that H2O2 may be involved in the capacitation process of human spermatozoa but not in the acrosome reaction.
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ABSTRACT: The present study examines the release of angiotensin‐converting enzyme (ACE) from human spermatozoa during capacitation conditions and in correlation to acrosome reaction and cell death. The ACE content of spermatozoa was measured by treating the cells with different detergents. Glass wool‐filtered and washed human spermatozoa were incubated for 3 hours at 37°C. Percentages of acrosome‐reacted and dead spermatozoa did not change significantly, but the ACE release increased from 0 to 2.93 ± 0.44 mU/100 × 10 6 spermatozoa after 180 minutes ( P < 0.001). In order to study the influence of acrosome reaction on ACE release, the acrosome reaction of noncapacitated spermatozoa was induced by 10 μM calcium ionophore A23187. The percentages of acrosome reaction and viability in noncapacitated spermatozoa as well as the ACE release were compared to corresponding data from experiments using capacitated spermatozoa (3 hours, 37°C) from the same donors. Although the number of living acrosome‐reacted spermatozoa after ionophore treatment (30.5 ± 4.0%) was significantly higher than after capacitation (13.3 ± 2.8%, P 0.001), ACE release from ionophore‐treated, noncapacitated spermatozoa was lower P < 0.05). The data indicate that ACE release from human spermatozoa during capacitation is independent of acrosome reaction. Measurement of ACE release may be a clinically useful assay for human sperm capacitation.
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Since the first endocannabinoid anandamide was identified in 1992, extensive research has been conducted to characterize the elements of the tightly controlled endocannabinoid signaling system. While it was established that the activity of endocannabinoids are terminated by a two-step process that includes cellular uptake and degradation, there is still a continuing debate about the mechanistic role of these processes in inactivating anandamide signals.
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Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca2+, and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 106 spermatozoa/ml), during which it transpired that the highest density (per mm2) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.
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In contrast to man and many other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. The apparent inability of stallion spermatozoa to penetrate the zona pellucida in vitro is most likely due to incomplete activation of spermatozoa (capacitation) because of inadequate capacitating or fertilizing media. In vivo , the oviduct and its secretions provide a microenvironment that does reliably support and regulate interaction between the gametes. This review focuses on equine sperm–oviduct interaction. Equine sperm–oviduct binding appears to be more complex than the presumed species-specific calcium-dependent lectin binding phenomenon; unfortunately, the nature of the interaction is not understood. Various capacitation-related events are induced to regulate sperm release from the oviduct epithelium and most data suggest that exposure to oviduct secretions triggers sperm capacitation in vivo . However, only limited information is available about equine oviduct secreted factors, and few have been identified. Another aspect of equine oviduct physiology relevant to capacitation is acid–base balance. In vitro , it has been demonstrated that stallion spermatozoa show tail-associated protein tyrosine phosphorylation after binding to oviduct epithelial cells containing alkaline secretory granules. In response to alkaline follicular fluid preparations (pH 7.9), stallion spermatozoa also show tail-associated protein tyrosine phosphorylation, hyperactivated motility and (limited) release from oviduct epithelial binding. However, these ‘capacitating conditions’ are not able to induce the acrosome reaction and fertilization. In conclusion, developing a defined capacitating medium to support successful equine IVF will depend on identifying as yet uncharacterized capacitation triggers present in the oviduct.
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