Search Results (352)

Plastic pollution represents a significant emerging environmental problem. Micro-sized particles of synthetic polymers—microplastics (MPs)—have been identified in all parts of marine ecosystems. In the marine environment, organisms are exposed to MPs, which undergo a constant process of physicochemical and biological degradation. Utilization of UV irradiation as the optimal exposure factor in the simulation of fundamental natural conditions is a widely accepted approach. This enables the study of the harmful effects of such particles when interacting with aquatic organisms. This study aimed to investigate the effect of pristine and photoaging primary polystyrene microspheres (µPS) at three concentrations on the viability and DNA integrity of the sperm of the sand dollars Scaphechinus mirabilis. The results of the investigation demonstrated that IR spectroscopy revealed structural changes in polystyrene, confirming the oxidative degradation of the polymer under UV irradiation. The study demonstrated that artificially aged µPS exhibited a more pronounced effect than pristine particles, as evidenced by reduced sperm viability and increased DNA damage. Thus, the resazurin test showed that after exposure to UV-irradiated µPS, sperm viability decreased to 83–85% at concentrations of 10 and 100 particles and to 70% at a concentration of 1000. In addition, the Comet assay showed that the particles increased the percentage of DNA in the tail from 20% to 30% in a dose-dependent manner. The findings substantiate and augment the existing body of experimental data of the toxicity of aged plastic fragments, thereby underscoring the need for further study into the toxicity of aged MPs on marine invertebrates. Full article

The roles of PIWI in mammalian spermatogenesis have been well-studied but are largely unknown in invertebrates such as the Chinese mitten crab (Eriocheir sinensis), which produces non-flagellar sperm. Here, we demonstrate that knockdown of PIWIs significantly promotes the proliferation of spermatogonia and the transformation into spermatocytes. Expression of PIWIs in HEK 293T significantly inhibits cell proliferation through the Wnt-signaling pathway. PIWIs suppress transcriptional activity of the Wnt pathway to down-regulate Cyclin D and Cyclin E by inhibiting β-catenin and the phosphorylation of β-catenin at Ser552. The intracellular structure of the adherens junction is destroyed by PIWIs due to downregulated α-catenin, β-catenin, and ZO1. Overall, our results suggest that PIWIs regulate spermatogonia self-renewal and differentiation through inhibiting the Wnt-signaling pathway and stabilize the structure of the adherens junction by regulating the expression and location of α-catenin, β-catenin, and ZO1 in E. sinensis, which are different from the functions in mammals. Our findings revealed novel functions and molecular mechanisms of PIWIs in regulating spermatogonia self-renewal and differentiation during the Crustacea spermatogenesis. Full article

Optimizing the cultivation system is crucial for tissue culture. The culture of cryopreserved testicular tissues is of great importance for the germplasm preservation of endangered animals and especially to ensure high-quality and high-output livestock. In this study, we compared two cultivation systems (Agarose-Supported system and Direct Adherent system) by evaluating their effects on tissue morphology, cell proliferation, apoptosis, gene expression, and endocrine function in cryopreserved testicular tissues from 30-day-old calves. The testicular tissues were cultured for 18 and 27 days with three biological replicates per group, aiming to identify which system better supports tissue preservation, cellular viability, and spermatogenic differentiation. This allowed us to clarify how different cultivation systems influence the structural maintenance and developmental potential of immature bovine testicular tissues. Histological and gene expression analyses revealed that the Agarose-Supported system better preserved the seminiferous cord architecture and supported the development of the seminiferous epithelium compared to the Direct Adherent system. The Agarose system significantly reduced the apoptosis and enhanced the expression of some key genes, including spermatogonial stem cell (SSC) markers (GFRα-1, UCHL1), meiotic marker (SYCP3), mature sperm marker (CRISP1), and testicular somatic cell markers (STAR, SOX9, ACTA2). The Agarose-Supported system also benefited spermatogenic differentiation and testosterone secretion. These findings demonstrate that the Agarose-Supported system facilitates the in vitro development of spermatogenic cells and Leydig cells in post-cryopreserved immature bovine testicular tissues. Full article

Male infertility affects nearly 15% of couples worldwide, with chromosomal abnormalities representing a major underlying cause. This review explores how numerical and structural chromosomal anomalies, along with environmental exposures, lifestyle factors, and age-related genetic changes, disrupt spermatogenesis and contribute to infertility. It synthesizes findings from cytogenetic, molecular, and clinical studies, with particular focus on mechanisms such as meiotic nondisjunction, spindle assembly checkpoint dysfunction, and alterations in cohesin and synaptonemal complex proteins. Chromosomal abnormalities, both numerical and structural, emerge as key contributors to male infertility by impairing chromosomal segregation and recombination, often leading to azoospermia or oligospermia. Meiotic checkpoint failures and recombination errors further exacerbate the production of aneuploid sperm. Environmental toxins, oxidative stress, and poor nutrition disrupt hormonal balance and chromatin integrity, while advancing paternal age is associated with increased sperm aneuploidy and impaired meiotic control, with implications for assisted reproduction. Specific syndromes, including AZF deletions, Kallmann syndrome, and 46,XX testicular DSD, exemplify the direct genetic impact on male fertility. Overall, chromosomal abnormalities are central to the pathophysiology of male infertility, arising from intrinsic meiotic errors as well as extrinsic environmental and lifestyle factors. Integrating cytogenetic diagnostics, genetic counseling, and lifestyle interventions is essential for comprehensive fertility assessment and management. Further research into molecular biomarkers and targeted therapies could enhance diagnosis, improve treatment strategies, and lead to better reproductive outcomes. Full article

Oxidative stress induces reactive oxygen species (ROS) accumulation, which compromises sperm DNA integrity, cellular homeostasis, and semen quality in Hainan black goats. This study aimed to mitigate ROS-mediated sperm damage by examining the protective effects of curcumin on metabolic regulation and sperm structural integrity. Semen samples were treated in vitro with varying concentrations of curcumin (5, 25, 50 μmol/L) under oxidative stress conditions. The intermediate concentration (25 μmol/L) was most effective at enhancing sperm quality. Following treatment, sperm motility, membrane integrity, and acrosome stability were significantly improved (p < 0.05), while ROS levels and apoptosis rates decreased. Antioxidant enzyme activities—glutathione peroxidase (GPX, p < 0.05), catalase (CAT, p < 0.05), and superoxide dismutase (SOD, p < 0.05)—were markedly elevated. Metabolomic analysis identified 48 differential metabolites (p < 0.05), including gluconic acid, 3-hydroxybutyric acid, and argininosuccinic acid, which were mainly involved in antioxidant defense, energy metabolism (e.g., the citrate cycle), and osmoregulatory pathways. Lipidomics revealed reduced lipid peroxidation and increased polyunsaturated fatty acid content, correlating with enhanced membrane stability. Transmission and scanning electron microscopy revealed preservation of sperm ultrastructure, with reduced mitochondrial and chromatin damage. Quantitative PCR further indicated curcumin-mediated downregulation of pro-apoptotic genes (BAX, Caspase3, and FAS) and upregulation of the anti-apoptotic gene BCL2 (p < 0.05). These findings demonstrate that Curcumin at 25 μM mitigated menadione-induced oxidative stress in goat sperm in vitro, improving antioxidant status, mitochondrial function and membrane integrity while reducing apoptosis. Multi-omic profiling supported redox and lipid homeostasis restoration. These findings establish proof-of-principle in an acute oxidative model. Full article

Arabinogalactan proteins (AGPs: hydroxyproline-rich glycoproteins) are ubiquitous in plants and play various functions in cases of development and reproduction. In Arabidopsis thaliana some AGPs can work as markers for gametophytic cell differentiation (among others embryological structures they mark generative cell wall and/or plasma membrane, and also sperm cells). However, apart from Arabidopsis, this labeling of generative cell and sperm cells in pollen grains has only been observed in a few flowering plant species belonging to dicotyledons. No such studies are available in monocotyledons. The main aim of our study was to see whether AGPs would be present at the generative cell–vegetative cell interface in different monocotyledons (representatives of Asparagaceae, Amarylidaceae and Liliaceae), and we also wanted to test whether they would be the same AGPs as in dicotyledons. For the study, we selected Gagea lutea (L.) Ker Gawl., Ornithogalum nutans L. and Galanthus nivalis L. species that differ in shape and size of generative cells. Antibodies against arabinogalactan proteins AGPs were used, including JIM8, JIM13, JIM14, MAC207, LM2, LM14, JIM15 and JIM4. The localization of the examined compounds was determined using immunohistochemistry techniques. The key finding was that AGPs (detected with JIM8 and JIM13 antibodies) consistently mark the boundary between the generative cell and the surrounding vegetative cytoplasm, suggesting their association with the generative cell–vegetative cell interface in all species studied. Identifying such molecular markers in male gametophyte may enhance the understanding of gametophytic cell fate, sperm cell identity and the molecular mechanisms underlying fertilization. Such labeling may also be useful in studies on pollen development, species comparisons, or responses to environmental stresses. Full article

Boar sperm is susceptible to damage by reactive oxygen species during in vitro preservation, leading to lipid peroxidation, which changes the sperm structure and affects its quality after thawing. Exogenous antioxidants play a vital role in preventing this damage. This research aimed to assess the impact of incorporating Mitoquinone into cryopreservation extenders on the quality and antioxidant capacity of boar sperm. Mitoquinone was added to the cryopreservation extender at varying concentrations, namely, 0, 50, 100, 150, and 200 nmol/L. Post-thawing, the sperm were examined for motility parameters, acrosome integrity, DNA integrity, mitochondrial activity, membrane integrity, and antioxidant enzyme activity. The results showed that compared with the control group, 150 nmol/L Mitoquinone could improve sperm viability after freezing and thawing and significantly reduce the malformation rate (p < 0.05). The addition of 150 nmol/L Mitoquinone led to a significant increase in the acrosome integrity, DNA integrity, mitochondrial activity, and membrane integrity of the boar sperm compared to the control group (p < 0.05). Moreover, it enhanced the antioxidant capacity of the sperm. This study demonstrated that the cryopreservation extender containing 150 nmol/L of Mitoquinone can enhance the effectiveness of semen cryopreservation. Full article

Bulls can develop various conditions affecting the penis, testicles, and associated structures that reduce fertility, spread disease, and increase costs for farmers. This review synthesizes data from farm fertility examinations and slaughterhouse findings to illustrate the prevalence of these conditions, their causes (including inherited defects, infections, and injuries), their impacts on breeding, and potential interventions. Conditions discussed include orchitis, testicular hypoplasia, testicular degeneration, testicular hydrocele, malignant tumors, spermatocele, penile papillomatosis, and penile hematoma. Lameness and systemic illnesses can also impair a bull’s breeding ability. Regular fertility examinations assessing overall health, testicle size, semen motility, and sperm morphology, coupled with timely medical or surgical treatment, can improve reproductive outcomes. Maintaining national records of birth defects and health histories could aid in preventing the transmission of deleterious traits. Reducing these disorders enhances animal welfare, protects herd fertility and the food supply, and supports the economic viability of cattle producers. Full article

Spermatogenesis in teleosts is essential for reproductive function; however, it varies considerably among species. The testis of the viviparous molly fish (Poecilia sphenops) was examined using both ultrastructural and immunohistochemical methods. The testis displays a restricted lobular type, where germ cells develop synchronously within Sertoli cell-forming cysts. Transmission electron microscopy (TEM) revealed all stages of spermatogenesis. Mature sperm are at the apex of the cysts and migrate toward the sperm ducts. Sperm duct epithelium is lined by cuboidal cells joined by tight junctions, with apical cilia and desmosomal complexes contributing to transport and structural integrity. The sperm ducts showed strong Periodic Acid–Schiff (PAS)-positive expression among negative stained spermatocysts. Centrally, a cavity serves as a storage area for spermatozoa that are organized into unencapsulated bundles known as spermatozeugmata. Sertoli cells exhibited extended cytoplasmic processes that supported developing germ cells, whereas Leydig cells occupied the interstitial tissue, contributing to hormonal regulation. Immunohistochemical labeling demonstrated strong vimentin expression in Sertoli cells and telocytes, indicating their mesenchymal origin and structural role. Calretinin expression was confined to Leydig cells and certain ductal epithelial cells, supporting its use as a marker for steroidogenic and secretory functions. These findings provide new insights into the testicular specialization of P. sphenops, highlighting key somatic–germ cell interactions, ductal adaptations, and marker expression patterns that underlie male reproductive success in viviparous fish. Full article

Background: Manganese (Mn) is a trace element essential for multiple physiological and biological processes. The testis plays a key role in male reproduction by producing sperm and synthesizing male hormones. This study investigates how Mn deficiency affects testicular development, spermatogenesis, and the blood–testis barrier (BTB), and evaluates associated variations in oxidative stress to explore potential mechanisms. Methods: A Mn-deficient diet was used to induce Mn deficiency in mice, with MnCl₂ administered via intraperitoneal injection. Mn levels in testicular tissue were measured by atomic absorption spectrometry. Testis and sperm morphology were assessed by H.E. and sperm staining. BTB markers were analyzed using immunofluorescence, Western blot, and qPCR. Oxidative stress was evaluated biochemically. Nrf2 pathway changes were detected by qPCR and Western blot. Results: The results indicated that Mn deficiency dramatically decreased the testicular index, caused abnormal testicular tissue structure, and significantly decreased Johnsen’s score. At the same time, sperm density and motility were significantly reduced, and the sperm deformity rate was significantly increased. In addition, the BTB function was impaired, as indicated by the significantly down-regulated expression of tight junction proteins including Occludin, ZO-1, JAM-A, and Claudin-11. As the oxidative stress levels increased, the mRNA and protein expression levels of molecules (including Nrf2 and HO-1) related to the Nrf2 signaling pathway were significantly down-regulated, while its inhibitor Keap1 exhibited significantly up-regulated expression. Notably, after supplementing MnCl₂, all the above abnormal indicators were significantly improved. Conclusions: Mn deficiency can lead to testicular tissue damage, decreased sperm quality, and BTB dysfunction, and the potential mechanism is probably closely associated with the increase in the oxidative stress level mediated by the Nrf2 pathway. Full article

Accurate assessment of sperm morphology is essential for artificial insemination using liquid-preserved boar semen. This study compared nine commonly used staining techniques, eosin, eosin–nigrosin, Diff-Quick^®, Hemacolor^®, Sangodiff-G^®, Spermac^®, Formol–Citrate–Rose Bengal stain, Testsimplets^®, and Methyl Violet, based on morphological assessment, cost, time efficiency, and storage stability. Each staining method was applied to 36 slides, totaling 324 samples, and evaluated four times each (1296 evaluations). Slides were analyzed four times: immediately after staining and after 1 day, 1 week, and 3 months of storage. The results indicated that Eosin was the fastest and most cost-effective method, providing strong contrast, though it increased detection of structural alterations. Eosin–nigrosin offered detailed morphology but formed colored crystals over time. Diff-Quick^® and Hemacolor^® showed good initial performance, but Hemacolor^® lost pigment clarity after 3 months (p = 0.0273). Sangodiff-G^® had poor contrast and reduced detection of abnormalities (p = 0.00229). Spermac^® delivered high contrast but was time-consuming. Formol–Citrate–Rose Bengal stain required extensive preparation and showed significant post-storage changes (p < 0.0001). Testsimplets^®, despite their high cost, suffered from declining interpretability (p < 0.0001). Methyl Violet lacked sufficient resolution and was highly unstable over time (p < 0.0001). In conclusion, Eosin emerged as the most practical and economical staining method for routine morphological evaluation of liquid-preserved boar semen. While eosin–nigrosin was also effective, its storage instability limits broader application. Other methods showed specific weaknesses, emphasizing the need to tailor stain selection to laboratory goals and constraints. Full article

The Mediterranean soft-shell turtle (Trionyx triunguis) is classified as critically endangered by the IUCN. Effective conservation requires a clear understanding of its reproductive strategies and population structure. By combining mitochondrial DNA tandem repeat-region profiling with genome-wide SNP data obtained through 3RADseq, we gained high-resolution insights into the genetic composition and breeding behavior of Mediterranean populations. Our results revealed complex reproductive dynamics, including multiple paternity, sperm storage, and repeated nesting within a single season—strategies that enhance genetic diversity in small, fragmented populations. Using SNP-based kinship inference, we estimated the number of breeding females and identified full and half-sibling groups, offering a robust genomic framework for assessing population size and structure. Genetic similarity patterns highlighted moderate differentiation among Israeli river populations, suggesting some connectivity, while samples from Türkiye were clearly distinct, reflecting long-term geographic and genetic separation. This integrative approach provides a scalable, repeatable tool for long-term monitoring. The combined use of maternal and biparental markers enables detailed tracking of genetic diversity, breeding contributions, and demographic trends—key elements for designing informed, adaptive conservation strategies. Full article

Interactions between protamines and DNA are essential for the correct structure of human sperm chromatin. Reproductive health can be adversely affected by environmental pollutants like per- and polyfluoroalkyl substances (PFAS). We previously reported that exposure to PFAS in the Veneto region causes alterations in sperm nuclear basic proteins (SNBP), along with reduced seminal antioxidant activity and increased lipoperoxides. This study analysed the protamine-to-histone ratio in SNBP and quantified the extent of DNA damage induced by SNBP in subjects in Veneto with serum perfluorooctanoic acid (PFOA) levels above the reference threshold. We found that all individuals with serum PFOA above the threshold exhibited grade three DNA damage, regardless of the protamine–histone ratio, which was generally altered but consistently shifted toward protamines. This indicate that exposure to PFAS can alter the protamine–histone ratio in these subjects. Moreover, SNBPs from these individuals showed reduced DNA-protective capacity under pro-oxidant conditions, suggesting a role in oxidative damage. To rationalize these effects, in this cross sectional study, we investigated the potential interactions between PFAS and human protamines by molecular docking analyses which showed that PFAS can form stable complexes with DNA through hydrophobic and polar interactions, especially with thymine pyrimidine rings. Further, docking analyses revealed that fluorine atoms in PFAS may interact with guanidinium groups in protamine P1 via electrostatic and van der Waals forces, competing with DNA for binding sites and potentially disrupting chromatin organisation. A ternary PFAS–DNA–protamine adduct may underpin the observed DNA damage. These results suggest that PFAS induce oxidative stress, which could affect male fertility. Full article

Introduction: Sperm morphology is a fundamental parameter in the evaluation of male infertility, offering critical insights into reproductive health. However, traditional manual assessments under microscopy are limited by operator dependency and subjective interpretation caused by biological variation. To overcome these limitations, there is a need for accurate and fully automated classification systems. Objectives: This study aims to develop a two-stage, fully automated sperm morphology classification framework that can accurately identify a wide spectrum of abnormalities. The framework is designed to reduce subjectivity, minimize misclassification between visually similar categories, and provide more reliable diagnostic support in reproductive healthcare. Methods: A novel two-stage deep learning-based framework is proposed utilizing images from three staining-specific versions of a comprehensive 18-class dataset. In the first stage, sperm images are categorized into two principal groups: (1) head and neck region abnormalities, and (2) normal morphology together with tail-related abnormalities. In the second stage, a customized ensemble model—integrating four distinct deep learning architectures, including DeepMind’s NFNet-F4 and vision transformer (ViT) variants—is employed for detailed abnormality classification. Unlike conventional majority voting, a structured multi-stage voting strategy is introduced to enhance decision reliability. Results: The proposed framework consistently outperforms single-model baselines, achieving accuracies of 69.43%, 71.34%, and 68.41% across the three staining protocols. These results correspond to a statistically significant 4.38% improvement over prior approaches in the literature. Moreover, the two-stage system substantially reduces misclassification among visually similar categories, demonstrating enhanced ability to detect subtle morphological variations. Conclusions: The proposed two-stage, ensemble-based framework provides a robust and accurate solution for automated sperm morphology classification. By combining hierarchical classification with structured decision fusion, the method advances beyond traditional and single-model approaches, offering a reliable and scalable tool for clinical decision-making in male fertility assessment. Full article

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. Full article