Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

Abstract

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.All isoforms of this enzyme have been described in human [17][18][19], bovine [20,21], murine [22] and boar [9] spermatozoa, among others. Furthermore, it has been demonstrated that NOS activity increases during in vitro capacitation in human sperm [5]. However, to the best of our knowledge, there are no previous studies identifying any NOS isoforms in ram spermatozoa.In mammals, melatonin is secreted by the pineal gland in response to light stimulus, and, apart from other functions, it modulates the secretion and synthesis of gonadotropic hormones via the hypothalamic-pituitary axis [23]. Thus, melatonin regulates reproductive seasonality in some species, such as ovine. Apart from pineal melatonin, this molecule is synthetized in other tissues and organs, including those in the male reproductive tract, as we recently demonstrated in ram [24]. Melatonin is also present in reproductive fluids such as seminal plasma [25] or follicular fluid [26]. This could explain how melatonin affects ram sperm functionality directly, modulating processes such as capacitation and decreasing apoptosic markers and oxidative stress [27]. The antioxidant capacity of melatonin in ram spermatozoa could be the reason for its antiapoptotic action, since high levels of reactive oxygen species (ROS) lead to cell death [28][29][30][31]. The ROS include superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), hydroxyl (OH) and nitric oxide (NO·) (also included in the reactive nitrogen species, RNS). All these molecules play an important role in sperm capacitation (reviewed by [32]).Melatonin can modulate NOS activity and, consequently, the nitric oxide in somat...

NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation

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