Search Results (228)

Infertility is a global health problem, with male factors contributing to nearly half of all cases. Up to 30% of male infertility is classified as idiopathic, in part because routine semen analysis does not assess sperm fertilizing competence. Capacitation is a complex process that endows spermatozoa with the competence to fertilize the oocyte, and it depends on oxidant-driven phosphorylation events. These events include increased PKA substrate and tyrosine phosphorylation, which promote hyperactivated motility and the acrosome reaction. These pathways are normally restrained by decapacitation factors that must be relieved in the female reproductive tract before capacitation can proceed. Protein CoAlation is an antioxidant modification of protein thiols through a disulfide bond with coenzyme A (CoASH). We previously detected protein CoAlation in human spermatozoa and observed that its levels decline during capacitation, but its function was unknown. We hypothesized that protein CoAlation functions as a decapacitation mechanism that prevents redox signalling, enabling oxidative activation of phosphorylation events during capacitation. Using spermatozoa from healthy human donors, we leveraged subcellular fractionation, immunocytochemistry, computer-assisted sperm analysis (CASA), and immunoblotting to determine the sperm protein CoAlation profile, assess CoASH biosynthetic enzymes, and test how pharmacological modulation of CoAlation levels influences capacitation. CoAlated proteins were distributed across intracellular sperm compartments, and spermatozoa possess the CoASH biosynthetic enzymes PANK2 and CoASY, indicating an intrinsic capacity for CoAlation. Inhibition of CoASH biosynthesis reduced CoAlation and enhanced PKA substrate phosphorylation, tyrosine phosphorylation, hyperactivated motility, and the progesterone-induced acrosome reaction under capacitating conditions. Pantothenic acid supplementation increased CoAlation and suppressed these processes without impairing viability or baseline motility. These findings indicate that high levels of protein CoAlation in several protein bands are a pre-existing feature of the non-capacitated state that restrains the redox-regulated events of capacitation and that its decline is required to permit sperm capacitation. CoAlation levels may emerge as a biomarker of sperm capacitation and fertilizing competence. Full article

Male infertility is an escalating global health issue, frequently associated with metabolic problems like obesity, diabetes, and age. Recent evidence designates AMP-activated protein kinase (AMPK) as a pivotal regulator linking energy balance to male reproductive function. AMPK regulates essential activities such as spermatogenesis, metabolic support of Sertoli cells, and steroidogenesis in Leydig cells, as well as sperm motility, capacitation, and the acrosome reaction. At the molecular level, AMPK coordinates signaling networks that include mTOR, SIRT1, PGC-1α, and FOXO to modulate mitochondrial function, oxidative stress, and autophagy-related quality control. Dysregulation of AMPK during metabolic and environmental stress results in compromised spermatogenesis, diminished sperm quality, mitochondrial malfunction, and reduced testosterone synthesis. Targeting AMPK signaling constitutes a possible therapeutic approach for enhancing male reproductive health. Pharmacological agents like metformin and AICAR, together with natural bioactive substances, lifestyle modifications, and exercise mimetics, have shown promise in reestablishing metabolic equilibrium and improving reproductive results. Moreover, combinatorial strategies that integrate antioxidants and autophagy modulators may yield synergistic advantages. Nonetheless, obstacles concerning tissue selectivity, optimum dose, and clinical translation persist. Future perspectives highlight precision medicine, biomarker-directed therapies, and the incorporation of metabolic health into fertility treatment. AMPK-targeted treatments collectively provide a novel and mechanistically sound method for addressing male infertility. Full article

Oxidative stress is one of the few defined causes of male infertility affecting at least one third of patients attending infertility clinics. Human spermatozoa are vulnerable to this form of attack because their stripped-down architecture means that they possess limited antioxidant protection and little capacity for biochemical repair. They also compound their vulnerability by being active generators of reactive oxygen species (ROS) and possessing multiple substrates for oxidative damage. The major sources of ROS in these cells are their mitochondria, an L-amino acid oxidase (IL4I1) and a calcium-dependent NADPH oxidase (NOX5). Spermatozoa tolerate the risks associated with ROS generation because their biology is heavily dependent on redox regulation. ROS are important mediators of sperm capacitation, stimulating the generation of cAMP and prostaglandins, inhibiting protein phosphatases and encouraging removal of cholesterol from the plasma membrane. Furthermore, during fertilization, the ability of ROS to activate metalloproteinases facilitates penetration of the zona pellucida and sperm–oocyte fusion. While ROS are physiologically important for sperm function, the over-production of these metabolites can impair sperm function. Antioxidants have therefore assumed some importance as a possible therapy for the infertile male. However, before this potential can be realized, we need to optimize the composition and dose of reagents used in such formulations and develop improved methods of diagnosing oxidative stress within the patient population. Full article

Nanotechnology, based on nanoparticles, has become an emerging interdisciplinary tool in reproductive biotechnology, offering innovative opportunities to improve fertilization efficiency and reproductive performance in farm animals. The purpose of this review is to provide an updated synthesis of current research on nanoparticle-based approaches that enhance in vitro fertilization outcomes and other assisted reproductive technologies. The focus is on the biological mechanisms, potential benefits, and limitations of nanoparticle use in animal reproduction. Nanoparticles—including gold, silver, zinc oxide, selenium, and magnetic iron oxide—exhibit distinctive physicochemical properties that enable targeted interactions with gametes and reproductive cells. When used in semen extenders or culture media, nanoparticles improve sperm motility, acrosome and membrane integrity, and reduce oxidative stress and apoptosis. These effects contribute to enhanced fertilization rates and higher embryo developmental competence. In addition, nanoparticles can function as carriers for hormones, antioxidants, and growth factors, stabilizing reagents essential for oocyte maturation, sperm capacitation, and early embryo culture. The review also discusses nanopurification (selectively isolating and removing particles) and nanosorting (separating or organizing nanoscale objects) techniques that allow for non-invasive selection of viable gametes, and fluorescence- and magnet-assisted sorting systems that increase precision in sperm sexing. The mechanical aspects of nanoparticle–cell interactions are analyzed, emphasizing the influence of particle size, dose, and surface modification on both biological efficacy and cytotoxicity. Safety, toxicological concerns, and regulatory frameworks—including International Organization for Standardization (ISO) standards and European Commission recommendations—are critically reviewed to highlight the need for harmonized biocompatibility criteria. Although nanoparticle use in animal reproduction remains largely experimental, accumulated evidence demonstrates its potential to improve reproductive efficiency and reduce economic losses. Integrating nanoparticle-based systems with existing reproduction platforms may represent a transformative step toward sustainable and precision-driven livestock breeding. Full article

This review provides a comparative examination of current methods for evaluating fertility and semen quality in mammals, with emphasis on livestock species. It is based on a structured search and analysis of 316 academic publications retrieved from major scientific databases, including Elsevier, Springer, Taylor & Francis, and MDPI, as well as other repositories, covering literature published between 1938 and 2025. This review examines advanced semen analysis techniques, including computer-assisted semen analysis (CASA) systems and assays for assessing chromatin integrity, DNA damage, seminal plasma composition, sperm membrane proteins, and functional parameters such as migration capacity, membrane integrity, acrosomal status, capacitation, morphology, viability, concentration, motility, and volume. Methods for semen collection are also outlined. The synthesis focuses on the comparative assessment of techniques and recent findings for major ruminant and monogastric livestock species: cattle (Bos taurus and Bos indicus), buffalo (Bubalus bubalis), sheep (Ovis aries), goats (Capra hircus), and pigs (Sus domesticus). A brief historical overview of assisted insemination is presented, incorporating relevant procedures and managerial factors that affect fertility outcomes. Advances in genetics related to reproductive health are also discussed. This synthesis seeks to integrate existing knowledge and guide future research in reproductive science for both animal production and human medicine. Full article

Na,K-ATPase (NKA) is expressed as two isoforms in mouse sperm: the testis-specific Na,K-ATPase α4 (NKAα4) and the somatic Na,K-ATPase α1 (NKAα1). Currently, the role that NKAα4 and NKAα1 play in the sperm acrosome reaction (AR) is unknown, and is the subject of our investigation. We studied the following: (1) the differential sensitivity of NKAα4 and NKAα1 to inhibition by ouabain; (2) the effects of deleting NKAα4 in mice (using NKAα4-KO mouse). In sperm from wild type (WT) mice, inhibiting NKAα4 with a low concentration of ouabain reduced AR. Inhibiting NKAα1 with a higher ouabain concentration further reduced AR, indicating that both NKA isoforms are necessary for AR. Surprisingly, sperm from NKAα4-KO mice exhibited an abnormally high AR. This was not due to a lack of acrosome development during sperm differentiation, but rather from premature release of the acrosome after they were isolated from the epididymis. When WT and NKAα4-KO sperm were exposed to media with or without Na⁺, K⁺, and Ca²⁺, or with the ionophores nigericin, valinomycin, and A23187, they displayed abnormal AR, indicating that NKAα4-KO sperm are unable to control intracellular Na⁺, K⁺, and Ca²⁺ levels. NKAα4 directly maintains intracellular Na⁺ and K⁺ and indirectly influences Ca²⁺ levels. This provides the necessary ion environment for full development of sperm AR. Along with NKAα4, NKAα1 also contributes to sperm AR. Full article

The yak represents a distinct domestic animal species that predominantly inhabits the Qinghai–Tibet Plateau and adjacent areas, possessing considerable value in both scientific and economic contexts. Compared to animals that mainly dwell on plains, such as cattle, the sperm maturation process in yak exhibits a certain degree of species specificity to adapt to their unique reproductive needs in high-altitude environments. Serving as the main storage site for functionally competent sperm, the cauda epididymis plays an integral role in mediating their post-testicular maturation. MiRNAs are vital regulatory molecules in the epididymis, influencing sperm maturation by modulating gene expression after transcription. To investigate the unique regulatory mechanisms of sperm maturation in yak, this study compared the miRNA expression profiles in the cauda epididymis of yak and cattle using high-throughput small RNA (sRNA) sequencing. The comparative analysis identified and characterized sRNA populations in the cauda epididymis of yak and cattle, revealing a similar length distribution that peaked at 22 nt and a predominance of known miRNAs. Notably, eight miRNAs were found to be highly expressed in both species. Furthermore, the first-nucleotide bias differed significantly between known and novel miRNAs within each species. A total of 31 differentially expressed (DE) miRNAs were identified, with 11 upregulated and 20 downregulated in yak compared to cattle. Among these, bta-miR-1298 exhibited the most significant upregulation, while bta-miR-2344 displayed the most pronounced downregulation. Bioinformatic analysis linked the predicted target genes of these miRNAs to numerous critical signaling pathways, including calcium signaling, the mitogen-activated protein kinase (MAPK) signaling pathway, the Ras-associated protein 1 (Rap1) signaling pathway, and the cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway. Furthermore, eight significantly DE miRNAs, including bta-miR-2443, bta-miR-503-3p, bta-miR-6517, bta-miR-2440, bta-miR-2431-3p, bta-miR-2436-3p, bta-miR-6523a, and bta-miR-6775, were predicted to target genes involved in various aspects of sperm structural and functional maturation. These aspects include flagellum formation, sperm motility, chromatin remodeling, acrosome reaction, acrosome structure, sperm capacitation, chemotaxis, and nuclear chromatin condensation. Multiple miRNAs and their corresponding predicted target genes were analyzed by quantitative real-time PCR (qPCR), demonstrating an inverse correlation between miRNA expression and target gene levels. These findings reveal a distinct, species-specific miRNA signature in the yak cauda epididymis, which suggests a potential contribution to regulating the epididymal luminal environment and the process of sperm maturation. This study provides preliminary foundational data for elucidating the differences in sperm maturation mechanisms between yak and cattle, and offers potential novel targets for improving reproductive efficiency in plateau livestock. Full article

Cannabis sativa, a species within the Cannabaceae family, produces a diverse range of phytochemicals, notably cannabinoids and terpenoids, with significant physiological and pharmacological relevance. Among its phytochemicals, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most studied for their psychoactive and medicinal properties. However, emerging evidence indicates that chronic or excessive exposure to these phytocannabinoids may adversely affect male fertility. This review synthesizes current knowledge on the influence of C. sativa and its constituents on the male reproductive system, with emphasis on spermatogenesis, sperm function, hormonal regulation, and the role of the endocannabinoid system (ECS). Experimental and clinical studies demonstrate that cannabinoids interact with CB1 and CB2 receptors expressed in the testes, epididymis, and spermatozoa, thereby modulating testosterone synthesis, sperm motility, morphology, and capacitation. THC, in particular, disrupts the hypothalamic–pituitary–gonadal (HPG) axis, leading to reduced luteinizing hormone and testosterone levels, impaired mitochondrial activity, and abnormal sperm morphology. Although CBD exhibits anti-inflammatory and antioxidant properties, its long-term impact on reproductive function remains uncertain. The review further highlights the complex interplay between exogenous cannabinoids and the endogenous ECS in maintaining reproductive homeostasis. Understanding these molecular mechanisms is critical for balancing the therapeutic potential of Cannabis-derived products with their reproductive risks. This knowledge could inform safe medicinal applications and contribute to the development of targeted cannabinoid-based therapies for male infertility. Full article

Background/Objectives: Male infertility, including primary and secondary infertility, is significantly influenced by oxidative stress, which disrupts sperm function and fertility. Seminal plasma, a protein-rich fluid essential for sperm protection and function, represents a valuable source for identifying biomarkers through proteomic analysis. While previous studies have explored seminal plasma proteins in fertility, the specific proteomic changes associated with oxidative stress in secondary infertility remain unclear. This study aimed to characterize these alterations by analyzing seminal plasma from three groups: men with secondary infertility, fertile donors with high oxidative stress, and fertile donors without oxidative stress. Methods: Pooled semen samples from each group underwent quantitative proteomics analysis using advanced mass spectrometry, with subsequent bioinformatic analysis using tools like DAVID, STRING, and IPA for identifying differentially expressed proteins (DEPs). Results: Quantitative proteomic analysis identified 377 DEPs in secondary infertility and 523 DEPs in fertile donors with high oxidative stress compared to controls. Bioinformatic analysis revealed seven shared pathways, including acute-phase response signaling, organismal injury, cellular movement, cell-to-cell signaling, free radical scavenging, immune cell trafficking, and Hematological system development. Notably, C3 and SERPINA3 exhibited significant alterations, along with proteins involved in sperm motility, capacitation, and fertilization, suggesting their potential roles in impaired fertility. Conclusions: These findings underscore the link between oxidative stress and secondary infertility and highlight specific seminal plasma proteins as potential biomarkers and therapeutic targets for diagnosing and treating male infertility. Full article

Conventional semen analysis fails to capture the molecular determinants underlying impaired reproductive function. Emerging evidence positions seminal plasma (SP) and extracellular vesicles (EVs) as dynamic regulators of sperm physiology, rather than passive transport components. SP, enriched with proteins, metabolites, hormones, and antioxidants, modulates sperm motility, capacitation, acrosome reaction, and immune tolerance. Complementarily, EVs, including prostasomes, epididymosomes, and testicular vesicles, deliver proteins, lipids, and small RNAs that remodel sperm membranes, protect against oxidative insults, and influence fertilization success. A critical dimension of the SP-EV axis is its role in balancing oxidative stress (OS) and endocrine signaling. Hormones and metabolic regulators within SP, together with EV-mediated transfer of receptors and regulatory RNAs, further integrate systemic metabolic health with local reproductive outcomes. Dysregulation of these networks, particularly in conditions such as varicocele, obesity, diabetes, and idiopathic infertility, compromises sperm function and reduces assisted reproductive technology (ART) success. This evidence-based review synthesizes current evidence on SP and EVs as ‘molecular gatekeepers’ in male infertility, emphasizing OS regulation, endocrine crosstalk, and their potential as biomarker reservoirs. By integrating proteomic, metabolomic, and transcriptomic insights, the translational opportunities for biomarker-informed diagnostics, prognostication, and therapeutic interventions are highlighted. Full article

Melamine, a nitrogen-rich industrial chemical, has raised increasing concern as an emerging environmental contaminant with potential reproductive toxicity. While its nephrotoxic effects are well established, the direct impact of melamine on human sperm remains poorly defined. In this study, we investigated the in vitro effects of melamine on human sperm, under both capacitating and non-capacitating conditions. Functional analyses revealed that the exposure to 0.8 mM melamine, the highest non-cytotoxic concentration in vitro, significantly compromised sperm motility and disrupted key capacitation processes, including tyrosine phosphorylation patterns, cholesterol efflux, and the acrosome reaction. Molecular assessments demonstrated melamine-induced mitochondrial dysfunction, characterized by COX4I1 downregulation, reduced mitochondrial membrane potential, and altered reactive oxygen species production. In parallel, gene expression analyses revealed the activation of apoptotic pathways, with the upregulation of BAX and downregulation of BCL2, changes that were more pronounced during capacitation. Furthermore, melamine exposure significantly increased sperm DNA fragmentation and denaturation, indicating genotoxic stress. Collectively, these findings demonstrate that even low, non-cytotoxic concentrations of melamine compromise sperm function by disrupting capacitation, mitochondrial activity, and genomic integrity. This study identifies capacitation as a critical window of vulnerability and underscores the need to consider melamine as a potential environmental risk factor for male reproductive health. Full article

Crucial regulators of gamete metabolism and signaling, mitochondria synchronize energy generation with redox equilibrium and developmental proficiency. Once thought of as hazardous byproducts, reactive oxygen species (ROS) are now understood to be vital signaling molecules that provide a “redox window of competence” that is required for oocyte maturation, sperm capacitation, and early embryo development. This review presents the idea of mitochondrial metabolic checkpoints, which are phases that govern gamete quality and fertilization potential by interacting with cellular signaling, redox balance, and mitochondrial activity. Recent research shows that oocytes may sustain a nearly ROS-free metabolic state by blocking specific respiratory-chain components, highlighting the importance of mitochondrial remodeling in gamete competence. Evidence from in vitro and in vivo studies shows that ROS act as dynamic gatekeepers at critical points in oogenesis, spermatogenesis, fertilization, and early embryogenesis. However, assisted reproductive technologies (ARTs) may inadvertently disrupt this redox–metabolic equilibrium. Potential translational benefits can be obtained via targeted techniques that optimize mitochondrial function, such as modifying oxygen tension, employing mitochondria-directed antioxidants like MitoQ and SS-31, and supplementing with nutraceuticals like melatonin, CoQ10, and resveratrol. Understanding ROS-mediated checkpoints forms the basis for developing biomarkers of gamete competence and precision therapies to improve ART outcomes. By highlighting mitochondria as both metabolic sensors and redox regulators, this review links fundamental mitochondrial biology to clinical reproductive medicine. Full article

Honey bees (Apis mellifera) are the main pollinators of many plant species, particularly agricultural crops. The concern over Colony Collapse Disorder of bee colonies in recent years necessitates the use of new approaches for their conservation in in situ and ex situ conditions. Modern techniques for cryopreservation of drone spermatozoa allow for the preservation of their genetic diversity. Some of the challenges in the field of cryopreservation are the alterations induced by the low temperatures, including morphological disruptions, plasma membrane integrity, formation of reactive oxygen species, DNA fragmentation, loss of motility, mitochondrial activity and viability, early hyperactivation, depletion of proteins from the acrosome region, premature capacitation, reduced sperm–oocyte fusion, and the occurrence of other cellular cryoinjuries. The objective of the current study is to contribute to the ongoing efforts in identifying substances added to semen extenders aimed at inhibiting cryogenic-induced changes. Our study investigates the impact of antioxidant supplements, scilicet vitamins C, vitamin E, and L-carnitine, on attenuating the adverse effects of cryogenic storage on drone spermatozoa. Using a Computer-Assisted Sperm Analysis, we evaluated the effectiveness of various antioxidants added to the extender in maintaining sperm motility parameters following liquid nitrogen storage. The data indicated significant differences in sperm traits among treatments with supplements after post-thawing. These findings emphasize the advantageous contribution of these added antioxidants within semen extenders for drone spermatozoa in preserving sperm quality parameters. The establishment of novel protocols for cryogenic storage of honey bee drone spermatozoa, incorporating low-cytotoxicity additives, is of utmost importance for the conservation of this endangered species. Full article

Autophagy is an intracellular process that recycles and degrades cytoplasmic components, including organelles and macromolecules, to provide energy and basic components for cell survival, maintain cellular homeostasis, and avoid self-damage. It is currently not fully known if mouse sperm undergoes the autophagy process, nor is the subcellular distribution, protein levels of autophagy-related proteins, and the biological role of autophagy in epididymal mouse sperm physiology fully understood. We aimed to investigate key autophagy markers, including LC3 (microtubule-associated protein 1A/1B-light chain 3), p62/SQSTM1 (Sequestosome 1), and mTOR (mechanistic Target of Rapamycin), in epididymal mouse sperm under capacitation (Cap) or non-capacitation (NC) conditions. Furthermore, we evaluated the possible role of these autophagy-related proteins on sperm viability, motility, intracellular pH (pHi), intracellular calcium concentrations [Ca²⁺]i, mitochondrial membrane potential, and acrosome reaction (AR) induction in the presence or absence of chloroquine (CQ), K67, and rapamycin. Our results suggest a dynamic re-localization of the autophagy-related proteins LC3, p62/SQSTM1, and mTOR under capacitation conditions. Moreover, treatment with specific autophagy inhibitors, such as CQ and K67, resulted in decreased LC3-II and p62/SQSTM1 protein levels. Additionally, rapamycin did not increase mTOR levels. Interestingly, treatment with these inhibitors also resulted in decreased motility, reduced mitochondrial membrane potential and hindered AR induction without affecting sperm viability. Overall, the presence and dynamic re-localization of LC3, p62/SQSTM1, and mTOR suggest that mouse epididymal sperm could perform initial steps of autophagy under capacitation conditions, and results of the pharmacological treatment could be associated with an important role of these autophagy-related proteins in sperm motility and AR induction. Full article

Cryopreservation of sperm is a resource used for artificial insemination. In the case of bovines, it ensures the reproduction of animals through cells from males with a highlighted genetic background. The fertilisation capacity of sperm cells is achieved after capacitation, a process that includes subcellular modifications that could be altered by cryopreservation. Intracellular calcium handling plays a crucial role in the development of capacitation, culminating in the acrosomal reaction. SERCA protein is responsible for calcium reuptake into the acrosome, which is one of the main calcium reservoirs of sperm cells. In this work, we studied the relationships between SERCA activity and sperm motility, capacitation progression, actin polymerisation, and intracellular calcium handling in cryopreserved bovine spermatozoa. Inhibition of SERCA activity reduced sperm motility and induced hyperactivation patterns. It also increased the proportion of cells with acrosomal reaction and earlier actin depolymerisation, an event necessary to induce the acrosomal reaction. All changes occurred in concordance with a significant increase in intracellular calcium concentration (Ca²⁺). Our findings suggest that a thapsigargin-sensitive Ca²⁺ pump consistent with SERCA activity remains responsive in cryopreserved spermatozoa from the bull studied, under specific cryopreservation and incubation conditions tested, and may contribute to Ca²⁺ handling, motility changes, and premature acrosomal exocytosis. Full article