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Leydig cells (LCs) play a pivotal role in male fertility, producing testosterone. Chromium (III) picolinate (CrPic 3 ), a contentious supplement with antidiabetic and antioxidant properties, raises concerns regarding male fertility. Using a rodent LC line, we investigated the cytotoxicity of increasing CrPic 3 doses. An insulin resistance (IR) model was established using palmitate (PA), and LCs were further exposed to CrPic 3 to assess its antioxidant/antidiabetic activities. An exometabolome analysis was performed using 1 H-NMR. Mitochondrial function and oxidative stress were evaluated via immunoblot. Steroidogenesis was assessed by quantifying androstenedione through ELISA. Our results uncover the toxic effects of CrPic 3 on LCs even at low doses under IR conditions. Furthermore, even under these IR conditions, CrPic 3 fails to enhance glucose consumption but restores the expression of mitochondrial complexes CII and CIII, alleviating oxidative stress in LCs. While baseline androgen production remained unaffected, CrPic 3 promoted androstenedione production in LCs in the presence of PA, suggesting that it promotes cholesterol conversion into androgenic intermediates in this context. This study highlights the need for caution with CrPic 3 even at lower doses. It provides valuable insights into the intricate factors influencing LCs metabolism and antioxidant defenses, shedding light on potential benefits and risks of CrPic 3 , particularly in IR conditions.

L-Carnitine, a natural antioxidant found in mammals, plays a crucial role in the transport of long-chain fatty acids across the inner mitochondrial membrane. It is used as a nutritional supplement by professional athletes, improving performance and post-exercise recovery. Additionally, its therapeutic applications, including those in male infertility, have been investigated, as it may act as a defense mechanism against the excessive production of reactive oxygen species (ROS) in the testis, a process that can lead to sperm damage. This effect is achieved by enhancing the expression and activity of enzymes with antioxidant properties. Nevertheless, the mechanisms underlying the benefits of L-Carnitine remain unknown. This review aims to consolidate the current knowledge about the potential benefits of L-Carnitine and its role in male (in)fertility. Considering in vitro studies with Sertoli cells, pre-clinical studies, and investigations involving infertile men, a comprehensive understanding of the effects of L-Carnitine has been established. In vitro studies suggest that L-Carnitine has a direct influence on somatic Sertoli cells, improving the development of germ cells. Overall, evidence supports that L-Carnitine can positively impact male fertility, even at a relatively low dose of 2 g/day. This supplementation enhances sperm parameters, regulates hormone levels, reduces ROS levels, and subsequently improves fertility rates. However, further research is needed to elucidate the underlying mechanisms and establish optimal doses. In conclusion, the role of L-Carnitine in the field of male reproductive health is highlighted, with the potential to improve sperm quality and fertility.

Low testosterone (T) levels are a major cause of male infertility, as this hormone is crucial for several processes throughout the entire male reproductive tract. Leydig cells (LC) produce T through testicular steroidogenesis. Disrupted LC function can hinder steroid production and fertility. Among the factors that affect steroidogenesis, endocrine-disrupting chemicals (EDCs) raise concerns, as they disturb hormonal signaling. Chromium is classified as an EDC, and its main forms are hexavalent (Cr(VI)) and trivalent chromium (Cr(III)). While Cr(III) is controversially regarded as an essential metal, its compound Cr(III) picolinate (CrPic 3 ) is used as a nutritional supplement due to its antidiabetic and antioxidant properties. This review aims to identify the possible effects of CrPic 3 on testicular steroidogenesis and thus, on male fertility. The detriments caused by CrPic 3 in LC include the inhibition of enzymes involved in steroidogenesis, and, as in other cells, the induction of mutagenesis and apoptosis. Remarkably, CrPic 3 impacts male fertility through the alteration of reactive oxygen species (ROS), T levels, and sperm parameters (sperm motility and abnormal sperm count). However, gaps and inconsistencies exist in the literature concerning its effects on male fertility. Thus, further research is imperative to comprehend the underlying mechanisms of CrPic 3 in the physiological processes relevant to male fertility, ensuring the supplement's safety for use by men.

Mitochondrial uncoupling proteins (UCPs) are central in the regulation of mitochondrial activity and reactive oxygen species (ROS) production. High oxidative stress is a major cause of male infertility; however, UCPs expression and function in human spermatozoa are still unknown. Herein, we aimed to assess the expression and function of the different homologs (UCP1-6) in human spermatozoa. For this purpose, we screened for the mRNA expression of all UCP homologs. Protein expression and immunolocalization of UCP1, UCP2, and UCP3 were also assessed. Highly motile spermatozoa were isolated from human normozoospermic seminal samples (n = 16) and incubated with genipin, an inhibitor of UCPs (0, 0.5, 5, and 50 µM) for 3 h at 37 °C. Viability and total motility were assessed. Mitochondrial membrane potential and ROS production were evaluated. Media were collected and the metabolic profile and antioxidant potential were analyzed by 1 H-NMR and FRAP, respectively. The expression of all UCP homologs ( UCP1-6 ) mRNA by human spermatozoa is herein reported for the first time. UCP1-3 are predominant at the head equatorial segment, whereas UCP1 and UCP2 are also expressed at the spermatozoa midpiece, where mitochondria are located. The inhibition of UCPs by 50 µM genipin, resulting in the UCP3 inhibition, did not compromise sperm cell viability but resulted in irreversible total motility loss that persisted despite washing or incubation with theophylline, a cAMP activator. These effects were associated with decreased mitochondrial membrane potential and lactate production. No differences concerning UCP3 expression, however, were observed in spermatozoa from normozoospermic versus asthenozoospermic men (n = 6). The inhibition of UCPs did not increase ROS production, possibly due to the decreased mitochondrial activity and genipin antioxidant properties. In sum, UCPs are major regulators of human spermatozoa motility and metabolism. The discovery and characterization of UCPs' role in human spermatozoa can shed new light on spermatozoa ROS-related pathways and bioenergetics physiology.

Aim : Calorie restriction (CR) diets and glucagon-Like Peptide-1 (GLP-1) analogs are known to alter energy homeostasis with the potential to affect the expression of obesity-related genes (ORGs). We hypothesized that CR and GLP-1 administration can alter ORGs expression in spermatozoa and testes, as well as the sperm parameters implicated in male fertility. Materials and Methods: Six-week-old adult male Wistar rats (n = 16) were divided into three groups, submitted either to CR (n = 6, fed with 30% less chow diet than the control rats), GLP-1 administration (n = 5, 3.5 pmol/min/kg intraperitoneal) for 28 days, or used as controls (n = 5, fed ad libitum). Selected ORGs expression, namely the fat mass and obesity-associated ( FTO ), melanocortin-4 receptor ( MC4R ), glucosamine-6-phosphate deaminase 2 ( GNPDA2 ), and transmembrane protein 18 ( TMEM18 ) were evaluated in testes and spermatozoa by a quantitative polymerase chain reaction (qPCR). Results: CR resulted in lower body weight gain and insulin resistance, but a higher percentage of sperm head defects. GLP-1 administration, despite showing no influence on body weight or glucose homeostasis, resulted in a lower percentage of sperm head defects. CR and GLP-1 administration were associated with a higher expression of all ORGs in the testes. Under CR conditions, the genes FTO and TMEM18 expression in the testes and the MC4R and TMEM18 transcripts abundance in sperm were positively correlated with the spermatozoa oxidative status. The abundance of FTO and TMEM18 in the spermatozoa of rats under CR were positively correlated with sperm concentration, while the testes' TMEM18 expression was also positively correlated with sperm vitality and negatively correlated with insulin resistance. Testes GNPDA2 expression was negatively correlated with sperm head defects. Conclusions: CR and GLP-1 administration results in higher ORGs expression in testes, and these were correlated with several alterations in sperm fertility parameters.

Stress, as a physiological response, is a major factor that affects several processes, including reproductive functions. The main hormonal players of stress are cortisol (humans) and corticosterone (rodents). Sertoli cells (SCs), as key contributors for the testicular homeostasis maintenance, are extensively challenged by different hormones, with glucocorticoid corticosterone being the signaling modulator that may impact these cells at different levels. We aimed to characterize how corticosterone modulates SCs energy balance, putting the mitochondrial performance and signaling output in perspective as the cells can disperse to the surroundings. TM4 mouse SCs were cultured in the absence and presence of corticosterone (in nM: 20, 200, and 2000). Cells were assessed for extracellular metabolic fluxes, mitochondrial performance (cell respirometry, mitochondrial potential, and mitochondrial complex expressions and activities), and the expression of androgen and corticosteroid receptors, as well as interleukine-6 (IL-6) and glutathione content. Corticosterone presented a biphasic impact on the extracellular fluxes of metabolites. Low sub-physiological corticosterone stimulated the glycolytic activity of SCs. Still, no alterations were perceived for lactate and alanine production. However, the lactate/alanine ratio was decreased in a dose-dependent mode, opposite to the mitochondrial complex II activity rise and concurrent with the decrease of IL-6 expression levels. Our results suggest that corticosterone finely tuned the energetic profile of mouse SCs, with sub-physiological concentrations promoting glycolytic expenditure, without translating into cell redox power and mitochondrial respiratory chain performance. Corticosterone deeply impacted the expression of the pro-inflammatory IL-6, which may alter cell-to-cell communication in the testis, in the last instance and impact of the spermatogenic performance., Competing Interests: The authors declare that they have no conflict of interest.

The incidence of metabolic diseases such as type 2 diabetes mellitus (DM) and obesity has been increasing dramatically. Both diseases are closely linked and new approaches for type 2 DM treatment aim to enable weight loss. A combined therapy of dapagliflozin and exenatide has been used against type 2 DM, influencing allbody glucose dynamics. Spermatogenesis is highly dependent on the metabolic cooperation established between Sertoli cells (SCs) and developing germ cells. To study the effects of dapagliflozin and exenatide on SC metabolism, mouse SCs were treated in the presence of sub-pharmacologic, pharmacologic, and supra-pharmacologic concentrations of dapagliflozin (50, 500, 5000 nM, respectively) and/or exenatide (2.5, 25, 250 pM, respectively). Cytotoxicity of these compounds was evaluated and the glycolytic profile, glycogen content assay, and lipid accumulation of SCs were determined. Dapagliflozin treatment decreased fat cellular deposits, demonstrating its anti-obesity properties at the cellular level. Polytherapy of exenatide plus dapagliflozin increased lactate production by SCs, which has been reported to improve sperm production and quality. Thus, the results herein suggest that the use of these two pharmacological agents can protect male fertility, while improving their glucose homeostasis and inducing weight loss.

Uncoupling proteins (UCPs) are transmembrane proteins members of the mitochondrial anion transporter family present in the mitochondrial inner membrane. Currently, six homologs have been identified (UCP1-6) in mammals, with ubiquitous tissue distribution and multiple physiological functions. UCPs are regulators of key events for cellular bioenergetic metabolism, such as membrane potential, metabolic efficiency, and energy dissipation also functioning as pivotal modulators of ROS production and general cellular redox state. UCPs can act as proton channels, leading to proton re-entry the mitochondrial matrix from the intermembrane space and thus collapsing the proton gradient and decreasing the membrane potential. Each homolog exhibits its specific functions, from thermogenesis to regulation of ROS production. The expression and function of UCPs are intimately linked to diabesity, with their dysregulation/dysfunction not only associated to diabesity onset, but also by exacerbating oxidative stress-related damage. Male infertility is one of the most overlooked diabesity-related comorbidities, where high oxidative stress takes a major role. In this review, we discuss in detail the expression and function of the different UCP homologs. In addition, the role of UCPs as key regulators of ROS production and redox homeostasis, as well as their influence on the pathophysiology of diabesity and potential role on diabesity-induced male infertility is debated.

Nowadays, infertility is classified as a disease of the reproductive system. Although it does not compromise the life of the individual, it can have detrimental effects on the physiological and psychological health of the couple. Male fertility evaluation is mainly focused on the analysis of sperm parameters. However, the ejaculated fluid is also composed of seminal plasma, and the study of this fluid can provide crucial information to help in the assessment of male fertility status. Total antioxidant capacity of the seminal plasma has been positively correlated with the fertility of men. Moreover, evidence highlights to a similar importance as that of female reproductive tract fluid antioxidant capabilities and female fertility. Herein, we describe the functions of seminal plasma and female reproductive tract fluids, as well as their main antioxidant components and their relationships with fertility outcomes. Additionally, this review contains the most up to date information regarding the mechanisms of the interaction between the male and the female reproductive fluids and the importance of proper antioxidant capacity for fertilization.

Male reproductive tissues are strongly susceptible to several environmental and lifestyle stressors. In general, male reproductive health is highly sensitive to oxidative stress, which results in reversible and/or irreversible changes in testosterone-producing cells, spermatogenesis, and sperm quality. Chromium compounds are widely used in the +3 and +6 valence states, as food supplements, and in the industrial field, respectively. Chromium (III) compounds, i.e., Cr(III)-tris-picolinate, [Cr(pic) 3 ], known as chromium picolinate, are used as nutritional supplements for the control of diabetes, body weight, and muscular growth. However, previous studies showed that animal models exposed to chromium picolinate experienced degenerative changes in spermatogenesis. Contradictory results are documented in the literature and deserve discussion. Furthermore, the long-term effects of chromium picolinate on the antioxidant system of treated subjects have not been properly studied. Comprehensive studies on the role of this compound will help to establish the safe and useful use of chromium supplementation. On the other hand, chromium (VI) compounds are widely used in several industries, despite being well-known environmental pollutants (i.e., welding fumes). Chromium (VI) is known for its deleterious effects on male reproductive health as toxic, carcinogenic, and mutagenic. Previous studies have demonstrated severe lesions to mouse spermatogenesis after exposure to chromium (VI). However, workers worldwide are still exposed to hexavalent chromium, particularly in electronics and military industries. Data from the literature pinpoints mechanisms of oxidative stress induced by chromium compounds in somatic and germ cells that lead to apoptosis, thus underlining the impairment of fertility potential. In this review, we analyze the benefits and risks of chromium compounds on male fertility, as well as the mechanisms underlying (in)fertility outcomes. Although supplements with antioxidant properties may maximize male fertility, adverse effects need to be investigated and discussed.

Acute pancreatitis (AP) is an inflammatory process of the pancreas with variable involvement of the pancreatic and peripancreatic tissues and remote organ systems. The main goal of this study was to evaluate the inflammatory biomarkers, oxidative stress (OS), and plasma metabolome of patients with different degrees of biliary AP severity to improve its prognosis. Twenty-nine patients with biliary AP and 11 healthy controls were enrolled in this study. We analyzed several inflammatory biomarkers, multifactorial scores, reactive oxygen species (ROS), antioxidants defenses, and the plasma metabolome of biliary AP and healthy controls. Hepcidin (1.00), CRP (0.94), and SIRI (0.87) were the most accurate serological biomarkers of AP severity. OS played a pivotal role in the initial phase of AP, with significant changes in ROS and antioxidant defenses relating to AP severity. Phenylalanine ( p < 0.05), threonine ( p < 0.05), and lipids ( p < 0.01) showed significant changes in AP severity. The role of hepcidin and SIRI were confirmed as new prognostic biomarkers of biliary AP. OS appears to have a role in the onset and progression of the AP process. Overall, this study identified several metabolites that may predict the onset and progression of biliary AP severity, constituting the first metabonomic study in the field of biliary AP.

Spermatozoa capacitation is a complex process that requires specific ionic and energetic conditions to support biochemical alterations leading to motility hyperactivation. However, human sperm capacitation is still poorly understood. Herein, we studied the effects of glucose on human sperm capacitation. Healthy men seminal samples ( n = 55) were submitted to a density gradient centrifugation and incubated in capacitating conditions in the absence or presence of increasing glucose concentrations (0, 5.5, 11, and 22 mM). Viability and total motility were accessed. Phosphotyrosine levels were measured. Mitochondrial activity and endogenous ROS production were evaluated. Oxidative stress-induced damage was analyzed. Culture media was collected and analyzed by 1 H-NMR. Our results show that glucose is essential for human sperm capacitation and motility. Notably, we observed that mitochondrial activity increased even in the absence of glucose. This increased mitochondrial activity was followed by a ROS overproduction, although no oxidative stress-induced damage was detected. Our results show that glucose is essential for capacitation but mitochondrial activation is independent from its stimuli. ROS overproduction may take part on a finely regulated signaling pathway that modulates or even activates capacitation. Taken together, our results constitute a paradigm shift on human sperm capacitation physiology.

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., Competing Interests: The authors declare no conflict of interest.