Alterations in nutrition and lifestyle patterns along the years have been associated with a decline in sperm quality and pregnancy rates. One of the main factors for this situation is the increasing prevalence of many diet-related health conditions and diseases with a negative impact on male reproductive potential, especially due to the generation of oxidative stress. In this chapter, we will discuss the impact of several nutritional factors in male reproduction and the possible use of antioxidant therapies to counteract the oxidative-induced damages.

BACKGROUND:Sertoli cells (SCs) provide essential metabolic support for spermatogenesis, including lactate production for developing germ cells. While insulin's role in SC glucose metabolism is well-established, its regulation of fatty acid (FA) uptake and oxidation-critical for germ cell support and residual body clearance-remains unknown. Insulin deficiency in diabetes mellitus (DM) impairs male fertility through mechanisms that may involve disrupted SC metabolism. OBJECTIVES:To determine whether insulin regulates FA uptake and oxidation in SCs and to elucidate mechanistic links between insulin deficiency and impaired spermatogenesis. MATERIALS AND METHODS:The TM4 mouse SC line was cultured under defined in vitro conditions in the presence of insulin (10 µg/mL) versus under insulin-deprived conditions for 18 h. FA metabolism was assessed using RT-qPCR (FAT/CD36, ACADL, and CPT1A), Western blotting, 1H-NMR spectroscopy, functional FA uptake assay, Oil Red O staining, JC-1 mitochondrial assessment, and Seahorse real-time metabolic analysis. RESULTS:Insulin deprivation significantly reduced glucose consumption, glutamine consumption, and lactate production. FAT/CD36 expression and FA uptake capacity decreased markedly, with reduced intracellular lipid droplet accumulation. Conversely, FA oxidation enzyme expression (CPT1A, ACADL) remained unchanged, as did mitochondrial membrane potential and oxygen consumption rates with/without etomoxir. DISCUSSION AND CONCLUSION:In the TM4 mouse SC line under defined in vitro conditions, insulin selectively regulates FA uptake while maintaining no control over downstream FA oxidation. This dissociation reveals that insulin modulates FA entry for storage and/or biosynthesis, while FA oxidation operates constitutively. Impaired FA uptake under insulin deficiency may compromise SC support for spermatogenesis, providing a mechanistic link to male infertility in DM that requires confirmation in primary SCs and in vivo diabetic models.

Male infertility affects 16.9% of couples and accounts for up to 30% of infertility cases. Its causes include genetic, acquired, and idiopathic factors, as well as environmental exposures and lifestyle habits like smoking and poor diet. Conventional semen analysis remains the gold standard but provides limited insight into molecular mechanisms and often fails to explain subfertility. Fertilization requires complex biochemical changes in spermatozoa, such as capacitation and the acrosome reaction, dependent on protein and glycoprotein interactions. Altered glycosylation patterns are linked to impaired capacitation, reduced fertilization potential, and disrupted spermatozoa-oocyte interactions. Specific glycoproteins, including clusterin, fibronectin, glycodelin-S, and others, are emerging as diagnostic and prognostic biomarkers. This review focuses on the structure, function, and glycosylation of glycoproteins in human semen, emphasizing their impact on male reproductive health and fertility. Their characterization offers new insights for understanding infertility and developing biomarkers for improved spermatozoa selection in assisted reproductive technologies.

l-Carnitine is a ubiquitous quaternary amine, essential for the mitochondrial β-oxidation of long-chain fatty acids, and a dietary supplement widely consumed by athletes due to its purported ergogenic and recovery-enhancing properties. Beyond its metabolic role in muscle tissue, l-Carnitine is highly concentrated in the male reproductive tract, suggesting a critical function in spermatogenesis. In the testis, Sertoli cells (SCs) act as essential "nurse cells," providing structural, nutritional, and immunomodulatory support to developing germ cells. This study elucidates the effects of l-Carnitine on SC viability, proliferation, redox state, and mitochondrial physiology, using an in vitro model (TM4 cell line) exposed to a range of l-Carnitine concentrations (0.005, 0.05, 0.5, and 5 mM) for 24 h. We demonstrate that a concentration mirroring plasma levels in athletes (0.05 mM) significantly enhances SC proliferation and metabolic viability, while selectively upregulating the expression of ATP synthase (Complex V). Conversely, supra-physiological doses (0.5 mM) induce a significant metabolic shift in the SC exometabolome, characterized by a marked reduction in the production of lactate, alanine, and acetate. This suggests a possible disruption of the metabolic support provided to germ cells. At the highest concentration (5 mM), l-Carnitine exhibits overt cytotoxicity, as evidenced by significant lactate dehydrogenase (LDH) leakage. Furthermore, l-Carnitine acts as a potent antioxidant, reducing endogenous reactive oxygen species (ROS) and mitigating oxidative damage to proteins and lipids. These findings provide mechanistic insights into how l-Carnitine use modulates the bioenergetic landscape of the seminiferous epithelium, suggesting that while physiological supplementation may bolster male fertility by improving SC health, excessive concentrations could paradoxically impair germ cell nutrition.

Anthocyanins are plant polyphenols widely regarded as antioxidants, yet they can exert concentration-dependent effects and act as pro-oxidants in specific contexts. Although their protective role in stressed testicular cells is established, their impact on Leydig cell steroidogenesis under non-pathological conditions remains poorly understood. Here, we investigated how an anthocyanin-enriched fraction from Callistemon citrinus (0-1.00 μg/mL) affects androgen synthesis in murine TM3 Leydig cells. Cell viability, intracellular ROS, antioxidant capacity, mitochondrial function, androstenedione production, steroidogenic gene expression, and the exometabolome by 1H-NMR were assessed. The fraction exhibited biphasic, dose-dependent effects. At 0.01 μg/mL, it induced a mitohormetic response, upregulating mitochondrial complexes III and V. Conversely, higher concentrations (0.10-1.00 μg/mL) reduced metabolic activity, increased intracellular ROS, and significantly suppressed androstenedione synthesis independently of Star. These concentrations also induced dose-dependent repression of Cyp17a1, concomitant with upregulation of Nr0b2, encoding the transcriptional repressor Small Heterodimer Partner (SHP). Overall, the data support a redox-dependent mechanism whereby elevated ROS promotes Nr0b2 expression, leading to Cyp17a1 suppression and impaired androstenedione production. These findings challenge the view of anthocyanins as uniformly beneficial for male fertility and identify Callistemon citrinus as a sustainable source of bioactive anthocyanins capable of modulating redox-endocrine homeostasis in a dose-dependent manner under basal conditions.

Over the years, caloric intake has remained a subject of profound scrutiny. Within the scientific community, there has been rigorous debate to ascertain which path is most ideal for enhancing quality of life and extending the human lifespan. Caloric restriction has been shown to be a promising contributor towards longevity and delaying the onset of age-related diseases. This diet consists of a reduction in caloric intake while maintaining essential energy and nutritional requirements to achieve optimal health while avoiding malnutrition. However, the effects of this nutritional regimen on male reproductive health have not yet been comprehensively studied. Nevertheless, such a complex process will certainly be regulated by a variety of metabolic sensors, likely sirtuins. Evidence has been gathered regarding this group of enzymes, and their ability to regulate processes such as chromatin condensation, the cell cycle, insulin signaling, and glucose and lipid metabolism, among many others. Concerning testicular function and male fertility, sirtuins can modulate certain metabolic processes through their interaction with the hypothalamic–pituitary–gonadal axis and mitochondrial dynamics, among many others, which remain largely unexplored. This review explores the impact of caloric restriction on male fertility, highlighting the emerging role of sirtuins as key regulators of male reproductive health through their influence on cellular metabolism.

Exposure to environmental chemicals, like pesticides, can impair male reproductive health by affecting Leydig cell (LC) function. This study investigated aminocarb, a widely used carbamate pesticide, and its impact on murine LCs (BLTK1 cell line). We observed a concentration-dependent biphasic toxicity, where higher aminocarb concentrations (500 μM) reduced LC viability, while lower doses (5 and 50 μM) unexpectedly increased it. Aminocarb selectively disrupted mitochondrial complexes I and III without altering overall mitochondrial respiration or reactive oxygen species (ROS) production. Notably, 50 μM aminocarb enhanced androstenedione production, a key androgen precursor. This research highlights the nuanced impact of aminocarb on LCs, suggesting targeted mitochondrial effects and steroidogenic stimulation, with implications for male reproductive health and environmental toxicology.

Male infertility affects 8–12% of couples worldwide and is solely responsible in up to 30% of cases. Assisted Reproductive Technologies (ARTs) provide potential solutions, particularly in conditions where spermatozoa display structural abnormalities or impaired motility, such as asthenozoospermia. Sperm metabolism demonstrates remarkable flexibility, shifting between glycolysis and oxidative phosphorylation to produce ATP required for motility. Glycerol kinase 2 (GK2) phosphorylates glycerol in the sperm midpiece, generating glycerol-3-phosphate, a key intermediate in glycolysis, lipid metabolism, and oxidative phosphorylation. The localization of GK2 suggests not only a regulatory role in sperm metabolism but also a possible association with VDAC proteins, contributing to ADP-ATP exchange between the cytosol and mitochondria. Elucidating the role of GK2 in spermatozoa is of particular relevance, as this enzyme not only contributes to key metabolic pathways but may also interact with VDAC proteins, influencing mitochondrial function and energy exchange. Such interactions could play a pivotal role in regulating sperm motility. A deeper understanding of these mechanisms could position GK2 as a valuable biomarker: in scenarios where GK2–VDAC interactions are confirmed, it may guide optimized sperm selection methods in ARTs, whereas the absence or impairment of such interactions could serve as a diagnostic indicator in asthenozoospermic men.

Leydig cells rely on lipids and fatty acids (FA) for essential functions like maintaining structural integrity, energy metabolism, and steroid hormone synthesis, including testosterone production. Carbamazepine (CBZ), a common anticonvulsant medication, can influence lipid metabolism and profiles, potentially impacting Leydig cell function and testosterone levels. Understanding this interplay is crucial to optimize treatment strategies for individuals requiring CBZ therapy while mitigating any adverse effects on male reproductive health. This study focuses on evaluating the effects of selected CBZ concentrations on the lipid homeostasis of BLTK-1 murine Leydig cells. By employing liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), we aimed to uncover the specific changes in lipid profiles induced by CBZ exposure (25 and 200 mu M). FA analysis demonstrated a significant decrease in FA 22:6 n-3 with increasing CBZ concentration and an increase in the n-6/n-3 ratio. Furthermore, changes in the lipidome, particularly in lipid species belonging to phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), and sphingomyelin (SM) classes were observed. PE and PC lipid species were significantly elevated in Leydig cells exposed to 200 mu M CBZ, whereas PG and SM species were downregulated. CBZ treatment significantly altered the Leydig cell phospholipidome, suggesting specific phospholipids such as PG 40:4, PG 34:1, PC O-32:1, PC 32:2, and PE P-38:6, which exhibited the lowest p-values, as potential biomarkers for clinical assessment of CBZ's impact on Leydig cells. These findings underscore the intricate relationship between CBZ exposure and alterations in lipid profiles, offering potential insights for monitoring and mitigating the drug's effects on male reproductive health.

Bisphenol A (BPA) is a persistent environmental pollutant harmful to male reproductive health. Although linked to testicular disorders, the underlying mechanisms and potential therapeutic agents to mitigate their adverse effects need to be fully explored. We investigated the impacts of perinatal BPA exposure and the therapeutic potential of β-caryophyllene (BCP) in the testes of adult Mongolian gerbils (Meriones unguiculatus). Pregnant females were exposed to a historically relevant and realistic dose of BPA (50 μg/kg/day) during gestation and lactation. The F1 male offspring were maintained until adulthood and received a 30-day treatment with BCP (50 mg/kg/day). We assessed the biometric, hormonal, sperm motility, oxidative markers, and metabolic testicular parameters. BPA-exposed gerbils showed decreased progressive sperm motility, as well as increased non-progressive sperm movement. ¹H-NMR metabolomics revealed testicular metabolic reprogramming, including deficits in central energy metabolism (e.g., acetate), nucleoside biosynthesis (adenosine, IMP), vitamin metabolism (pyridoxine, taurine), and phospholipid integrity (ethanolamine, O-phosphoethanolamine). Pathway analysis revealed disturbances in pyruvate metabolism/gluconeogenesis, purine metabolism, and glycerophospholipid metabolism, confirming altered testicular homeostasis. Notably, the BCP consumption rescues sperm motility and mitigates BPA-induced metabolic dysfunction by restoring testicular contents of acetate, adenosine, inosine, IMP, ethanolamine, and O-phosphoethanolamine. Perinatal exposure to BPA programs latent testicular dysfunction through the establishment of a toxic metabolic memory that manifests in adulthood. However, the 30-day BCP intervention mitigated BPA-induced testicular metabolic dysfunctions. This study provides novel descriptive insights into BPA-induced testicular toxicity and highlights BCP as a promising therapeutic agent to counteract the long-term reproductive consequences of early-life exposures.

Exposure to pesticides, poses a significant threat to male fertility by compromising crucial cells involved in spermatogenesis. Aminocarb, is a widely used carbamate insecticide, although its detrimental effects on the male reproductive system, especially on sustentacular Sertoli cells, pivotal for spermatogenesis, remains poorly understood. In this study, we investigated the effects of escalating concentrations of aminocarb on a mouse Sertoli cell line, TM4. Assessments included cytotoxic analysis, mitochondrial biogenesis and membrane potential, expression of apoptotic proteins, caspase-3 activity, and oxidative stress evaluation. Our findings revealed a dose-dependent reduction in the proliferation and viability of TM4 cells following exposure to increasing concentrations of aminocarb. Notably, exposure to 5 μM of aminocarb induced depolarization of mitochondria membrane potential, and a significant decrease in the ratio of phosphorylated eIF2α to total eIF2α, suggesting heightened endoplasmic reticulum stress via the activation of the eIF2α pathway. Moreover, the same aminocarb concentration was demonstrated to increase both caspase-3 protein levels and activity, indicating an apoptotic induction. Collectively, our results demonstrate that aminocarb serves as an apoptotic inducer for mouse sustentacular Sertoli cells in vitro, suggesting its potential to modulate independent pathways of the apoptotic cascade. These findings underscore the deleterious impact of aminocarb on spermatogenic performance and male fertility, highlighting the urgent need for further investigation into its mechanisms of action and mitigation strategies to safeguard male fertility.

Leydig cells (LCs) play a pivotal role in male fertility, producing testosterone. Chromium (III) picolinate (CrPic3), a contentious supplement with antidiabetic and antioxidant properties, raises concerns regarding male fertility. Using a rodent LC line, we investigated the cytotoxicity of increasing CrPic3 doses. An insulin resistance (IR) model was established using palmitate (PA), and LCs were further exposed to CrPic3 to assess its antioxidant/antidiabetic activities. An exometabolome analysis was performed using 1H-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 CrPic3 on LCs even at low doses under IR conditions. Furthermore, even under these IR conditions, CrPic3 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, CrPic3 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 CrPic3 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 CrPic3, particularly in IR conditions.

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 (CrPic3) is used as a nutritional supplement due to its antidiabetic and antioxidant properties. This review aims to identify the possible effects of CrPic3 on testicular steroidogenesis and thus, on male fertility. The detriments caused by CrPic3 in LC include the inhibition of enzymes involved in steroidogenesis, and, as in other cells, the induction of mutagenesis and apoptosis. Remarkably, CrPic3 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 CrPic3 in the physiological processes relevant to male fertility, ensuring the supplement's safety for use by men.

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.

IntroductionFertility rates in developing countries have declined over the past decades, and the trend of delayed fatherhood is rising as societies develop. The reasons behind the decline in male fertility with advancing age remain mysterious, making it a compelling and crucial area for further research. However, the limited number of studies dedicated to unraveling this enigma poses a challenge. Thus, our objective is to illuminate some of the upregulated and downregulated mechanisms in the male testis during the aging process.Areas coveredHerein, we present a critical overview of the studies addressing the alterations of testicular proteome through the aging process, starting from sexually matured young males to end-of-life-expectancy aged males. The comparative studies of the proteomic testicular profile of men with and without spermatogenic impairment are also discussed and key proteins and pathways involved are highlighted.Expert opinionThe difficulty of making age-comparative studies, especially of advanced-age study subjects, makes this topic of study quite challenging. Another topic worth mentioning is the heterogeneous nature and vast cellular composition of testicular tissue, which makes proteome data interpretation tricky. The cell type sorting and comorbidities testing in the testicular tissue of the studied subjects would help mitigate these problems.

Aging is associated with testicular morphological and functional alterations, but the underlying molecular mechanisms and the impact of physical exercise are poorly understood. In this study, we examined the effects of age and lifelong moderate-intensity exercise on rat testis. Mature adults (35 weeks) and middle-aged (61 weeks) Wistar Unilever male rats were maintained as sedentary or subjected to a lifelong moderate-intensity treadmill training protocol. Testis weight and histology, mitochondrial biogenesis and function, and proteins involved in protein synthesis and stress response were evaluated. Our results illustrate an age-induced testicular atrophy that was associated with alterations in stress response, and mitochondrial biogenesis and function. Aging was associated with increased testicular levels of heat shock protein beta-1 (HSP27) and antioxidant enzymes. Aging was also associated with decreased mRNA abundance of the nuclear respiratory factor 1 (Nrf1), a key transcription factor for mitochondrial biogenesis, which was accompanied by decreased protein levels of the oxidative phosphorylation system (OXPHOS) complexes subunits in the testes of older animals. On the other hand, exercise did not protect against age-induced testicular atrophy and led to deleterious effects on sperm morphology. Exercise led to an even more pronounced decrease in the Nrf1 mRNA levels in testes of both age groups and was associated with decreased mRNA abundance of other mitochondrial biogenesis markers and decreased protein levels of OXPHOS complexes subunits. Lifelong moderate-intensity exercise training was also associated with an increase in testicular oxidative stress markers and possibly with reduced translation. Together, our results indicate that exercise did not protect against age-induced testicular atrophy and was not associated with beneficial changes in mitochondria and stress response, further activating mechanisms of protein synthesis inhibition.

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.

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.

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.