Spermatozoa are physiologically exposed to reactive oxygen species (ROS) that play a pivotal role on several sperm functions through activation of different intracellular mechanisms involved in physiological functions such as sperm capacitation associated-events. However, ROS overproduction depletes sperm antioxidant system, which leads to a condition of oxidative stress (OS). Subfertile and infertile men are known to present higher amount of ROS in the reproductive tract which causes sperm DNA damage and results in lower fertility and pregnancy rates. Thus, there is a growing number of couples seeking fertility treatment and assisted reproductive technologies (ART) due to OS-related problems in the male partner. Interestingly, although ART can be successfully used, it is also related with an increase in ROS production. This has led to a debate if antioxidants should be proposed as part of a fertility treatment in an attempt to decrease non-physiological elevated levels of ROS. However, the rationale behind oral antioxidants intake and positive effects on male reproduction outcome is only supported by few studies. In addition, it is unclear whether negative effects may arise from oral antioxidants intake. Although there are some contrasting reports, oral consumption of compounds with antioxidant activity appears to improve sperm parameters, such as motility and concentration, and decrease DNA damage, but there is not sufficient evidence that fertility rates and live birth really improve after antioxidants intake. Moreover, it depends on the type of antioxidants, treatment duration, and even the diagnostics of the man’s fertility, among other factors. Literature also suggests that the main advantage of antioxidant therapy is to extend sperm preservation to be used during ART. Herein, we discuss ROS production and its relevance in male fertility and antioxidant therapy with focus on molecular mechanisms and clinical evidence.

Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support capacitation. While this increased energy demand during capacitation is well established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.

AMP-activated protein kinase AMPK regulates cellular energy by controlling metabolism through the inhibition of anabolic pathways and the simultaneous stimulation of catabolic pathways. Given its central regulator role in cell metabolism, AMPK activity and its regulation have been the focus of relevant investigations, although only a few studies have focused on the AMPK function in the control of spermatozoa’s ability to fertilize. This review summarizes the known cellular roles of AMPK that have been identified in mammalian spermatozoa. The involvement of AMPK activity is described in terms of the main physiological functions of mature spermatozoa, particularly in the regulation of suitable sperm motility adapted to the fluctuating extracellular medium, maintenance of the integrity of sperm membranes, and the mitochondrial membrane potential. In addition, the intracellular signaling pathways leading to AMPK activation in mammalian spermatozoa are reviewed. We also discuss the role of AMPK in assisted reproduction techniques, particularly during semen cryopreservation and preservation (at 17 °C). Finally, we reinforce the idea of AMPK as a key signaling kinase in spermatozoa that acts as an essential linker/bridge between metabolism energy and sperm’s ability to fertilize.

Mammalian sperm must undergo capacitation as a preparation for entering into hyperactivated motility, undergoing the acrosome reaction, and acquiring fertilizing ability. One of the initial capacitation events occurs when sperm encounter an elevated HCO₃⁻ concentration. This anion activates the atypical adenylyl cyclase Adcy10, increases intracellular cAMP, and stimulates protein kinase A (PKA). Moreover, an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is essential for sperm capacitation. Although a cross-talk between cAMP-dependent pathways and Ca²⁺ clearly plays an essential role in sperm capacitation, the connection between these signaling events is incompletely understood. Here, using three different approaches, we found that CatSper, the main sperm Ca²⁺ channel characterized to date, is up-regulated by a cAMP-dependent activation of PKA in mouse sperm. First, HCO₃⁻ and the PKA-activating permeable compound 8-Br-cAMP induced an increase in [Ca²⁺]_i, which was blocked by the PKA peptide inhibitor PKI, and H89, another PKA inhibitor, also abrogated the 8-Br-cAMP response. Second, HCO₃⁻ increased the membrane depolarization induced upon divalent cation removal by promoting influx of monovalent cations through CatSper channels, which was inhibited by PKI, H89, and the CatSper blocker HC-056456. Third, electrophysiological patch clamp, whole-cell recordings revealed that CatSper activity is up-regulated by HCO₃⁻ and by direct cAMP injection through the patch-clamp pipette. The activation by HCO₃⁻ and cAMP was also blocked by PKI, H89, Rp-cAMPS, and HC-056456, and electrophysiological recordings in sperm from CatSper-KO mice confirmed CatSper's role in these activation modes. Our results strongly suggest that PKA-dependent phosphorylation regulates [Ca²⁺]_i homeostasis by activating CatSper channel complexes.

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca²⁺ and bicarbonate TCM, time from 1–24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca²⁺ and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.