Search Results (42)

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

Background/Objectives: Testicular dysfunction is a side effect of radiotherapy due to off-target damage. Germ cells are highly vulnerable. Although Sertoli and Leydig cells are more resistant, they are still affected, impairing spermatogenesis and steroidogenesis. With rising youth cancer rates, strategies to preserve fertility are crucial. Losartan (LOS) has potential to mitigate this damage. This work aimed to determine acute and late effects of radiotherapy in testicular metabolism and if LOS mitigates those effects. Methods: Male Wistar rats (n = 47, 12 weeks old) received 2.5 Gy of ionizing radiation to the scrotum (1.05 Gy/min). LOS-treated rats received 34 mg/kg twice daily before, during and after irradiation. Animals were euthanized at 2 and 60 days post-exposure, to represent acute and late effects, respectively. Reproductive organs were weighed, serum hormones assessed (ELISA), testicular mRNA expression quantified (qPCR) and oxidative stress markers, such as lipid peroxidation, protein carbonylation, and protein nitration measured (slot-blot). Metabolomic profiles were obtained via ¹H-NMR. Results: Acute irradiation reduced seminal vesicle weight, increased FSH, and decreased sperm concentration. Late effects included reduced testicular and epididymal weight, impaired sperm quality, increased protein carbonylation, and altered metabolic profiles. LOS mitigated acute weight loss but not sperm decline. Long-term, LOS improved sperm quality, reduced oxidative stress, and promoted adaptive metabolic responses. Conclusions: Irradiation-based cancer therapy causes structural and functional testicular damage and changes the testicular metabolome of rats, while LOS has the potential to be used as a radioprotector to mitigate the adverse acute and late effects of radiation on male fertility. Full article

Background/Objectives: Approximately 50% of infertility cases are attributable to male factors; yet conventional semen examination can not identify the molecular abnormalities that hinder sperm functionality. Extracellular vesicles (EVs) derived from sperm, such as testicular EVs, prostasomes, and epididymosomes, have become important modulators of oocyte activation, sperm maturation, capacitation, acrosome stability, motility, and early embryonic development. This study aimed to evaluate the potential diagnostic and translational uses of sperm-associated extracellular vesicles (EVs) in male infertility and assisted reproduction, while also consolidating recent insights on their origins, composition, and functional significance. Methods: A focused narrative search of PubMed (2000–2025) was conducted using backward and forward citation tracking. Studies that qualified included human clinical cohorts, functional sperm extracellular vesicle tests, and omics analyses using MISEV-aligned extracellular vesicle isolation and characterisation methodologies. When human mechanistic understanding was constrained, knowledge from animal research was selectively integrated. Results: The cargo signatures specific to the source identified in sperm-derived and seminal EVs encompass proteins, small RNAs, lipids, and enzymatic modules that govern sperm maturation, capacitation, acrosome reaction, redox balance, calcium signalling, zona binding, and DNA integrity. Density-resolved seminal extracellular vesicle subfractions (EV-H/EV-M/EV-L) have unique functional and proteomic characteristics linked to progesterone-induced hyperactivation, oxidative stress, and motility. Asthenozoospermia and oligoasthenoteratozoospermia are associated with changes in extracellular vesicle composition, reduced embryonic developmental potential, compromised oocyte activation (related to PLCζ), and increased sperm DNA fragmentation. Numerous EV-related miRNA and protein signatures may predict TESE results, identify functional sperm anomalies not recognised by conventional semen analysis, and differentiate between obstructive and non-obstructive azoospermia. Conclusions: The available findings indicate that sperm-derived extracellular vesicles are significant functional regulators of sperm physiology and may serve as valuable non-invasive indicators for male infertility. The standardisation of EV isolation, characterisation, and clinical validation is essential prior to widespread use; nonetheless, their integration into liquid biopsy methods and assisted reproductive technology processes represents a significant improvement. Full article

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. Identifying proteins associated with EPs that regulate host responses to physiological and pathophysiological processes is of critical importance. Here, we report the findings of our study to gain insight into the proteins associated with NVEPs. We used samples from human semen, the rat brain, and the rhesus macaque (RM) brain and blood to assess the physical properties and proteome profiles of NVEPs from these specimens. The results show significant differences in the zeta potential, concentration, and size of NVEPs across different species. We identified 938, 51, and 509 total proteins from NVEPs isolated from rat brain tissues, RM blood, and human seminal plasma, respectively. The species-specific protein networks show distinct biological themes, while the species-conserved protein interactome was identified with six proteins (ALB, CST3, FIBA/FGA, GSTP1, PLMN/PLG, PPIA) associated with NVEPs in all samples. The six NVEP-associated proteins are prone to aggregation and formation of wide, insoluble, unbranched filaments with a cross-beta sheet quaternary structure, such as amyloid fibrils. Protein-to-function analysis indicates that the six identified proteins are linked to the release of dopamine, immune-mediated inflammatory disease, replication of RNA viruses, HIV-HCV co-infection, and inflammation. These interesting findings have created an opportunity to evaluate NVEPs for their potential use as biomarkers of health and disease. Additional in-depth studies are needed to clarify when and how these proteins sustain their physiological role or transition to pathogenic roles. Full article

Background: Extracellular vesicles, particularly exosomes, play a crucial role in cell–cell communication and as carriers of biomarkers. However, their use in clinical settings is limited due to a lack of standardized isolation and characterization. Ultracentrifugation (UC) is considered a gold standard for exosome isolation but presents several limitations. Size-exclusion chromatography (SEC) has recently gained attention as a superior method, which offers better yield, purity, and protection of exosome physical properties. This study focused on optimizing the SEC method for isolation of exosomes from seminal plasma and comparing yield, quality, and proteome profiles with those obtained by UC. Methods: In this SEC method, seminal plasma (0.5 mL) was loaded onto a SEC column and collected in 13 fractions of 0.4 mL each. The physical and molecular characterization of exosomes was carried out using a ZetaView analyzer and Western blot, respectively. Further, SEC-isolated exosomes were used for proteomic profiling and functional bioinformatic analysis. Results: The second and third fractions had the highest concentration of exosomes with uniform size and strong expression of exosome markers. Also, comparative proteomic analysis identified 3315 proteins in SEC-isolated exosomes and 931 in UC-isolated exosomes, with 709 proteins in common. SEC-isolated exosomes showed greater overlap with Vesiclepedia’s and ExoCarta’s top 100 lists than UC-isolated exosomes (Vesiclepedia: 91 vs. 77 proteins, ExoCarta: 94 vs. 79). Proteins from SEC- and UC-isolated exosomes showed similar enrichment profiles across all three gene ontology categories. Conclusions: Overall, this optimized SEC protocol is a reliable alternative method to isolate seminal exosomes with high purity, supporting its potential applications in clinical and basic research. Full article

In this study, oyster fermentation broth (OFB) was prepared by fermenting oysters with yeast, and its effects on oxidative stress and reproductive damage induced by tripterygium glycosides (TG) in male rats were investigated. Component analysis revealed that OFB contained bioactive substances including proteins (1.19 g/L), taurine (0.76 g/L), organic acids (2.30 mg/mL), polyphenols (123.00 mg GAE/L), flavonoids (1.97 mg RE/L), and zinc (1.10 mg/L). In vitro study revealed that OFB exhibited notable antioxidant activity, with a total antioxidant capacity of 1.28 U/mL, and DPPH, ABTS, and hydroxyl radical scavenging rates of 55.80%, 69.54%, and 48.36%, respectively. Animal experiments showed that, compared with the TG-induced model group, rats administered both low-dose (5 mL/kg) and high-dose (10 mL/kg) OFB showed significantly increased testis and seminal vesicle + prostate indices, sperm count, and serum testosterone (T) levels and decreased sperm malformation rate (p < 0.01 for all). Histological analysis of the testis revealed an increased number of spermatogenic cells and sperm within the seminiferous tubules, along with ameliorated pathological conditions compared to the model group. Potential mechanisms might be related to OFB increasing the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) enzymes and reducing levels of malondialdehyde (MDA) in testis (p < 0.01). The findings demonstrated that OFB successfully alleviated TG-induced reproductive damage in male rats, which might be attributed to its excellent antioxidant effect. The study offers valuable insights for producing functional foods from oysters and further validates OFB’s efficacy in promoting reproductive function. Full article

Honey bees (Apis mellifera ligustica) are essential pollinators in both ecosystems and agricultural production. However, their populations are declining due to various factors, including pesticide exposure. Despite their importance, the reproductive castes, particularly drones, remain understudied in terms of pesticide effects. To investigate the effects of azoxystrobin and picoxystrobin on honey bee drones, the drones were exposed to different concentrations of azoxystrobin and picoxystrobin for 14 days; the drone survival, body weight, nutrient content, reproductive organs, and sperm concentration were assessed. Results showed that exposure to both fungicides caused a significant reduction in drone survival rates, with survival rates decreasing progressively as the duration of exposure increased. Compared to the control group, the body weights of drones in all treatment groups were significantly lower on days 7 and 14. Nutrient analysis revealed that low concentrations of azoxystrobin and picoxystrobin increased protein levels, while free fatty acid content decreased significantly in all treatment groups. No significant changes were observed in the total carbohydrate content. Morphological examination of reproductive organs showed that the lengths of the mucus glands and seminal vesicles in drones were significantly shorter in the treatment groups compared to the control group. Furthermore, exposure to azoxystrobin and picoxystrobin resulted in a significant decline in sperm concentration in the drones. These findings indicate that azoxystrobin and picoxystrobin have adverse effects on the health and reproductive capacity of honey bee drones. The present study highlights the need to reassess the risks posed by these fungicides to pollinators, particularly given the critical role of drones in maintaining the genetic diversity and resilience of honey bee colonies. Further research is warranted to elucidate the underlying mechanisms of these effects and explore potential mitigation strategies. Full article

Artificial insemination (AI) is a key breeding technique in the swine industry; however, the lack of reliable biomarkers for semen quality limits its effectiveness. Seminal plasma (SP) contains extracellular vesicles (EVs) that present a promising, non-invasive biomarker for semen quality. This study explores the biochemical profiles of boar SP to assess semen quality through near-infrared spectroscopy (NIRS) and proteomics of SP-EVs. Fresh semen from mature Duroc boars was evaluated based on sperm motility, classifying samples as Passed (≥70%) or Failed (<70%). NIRS analysis identified distinct variations in water structures at specific wavelengths (C1, C5, C12 nm), achieving high accuracy (92.2%), sensitivity (94.2%), and specificity (90.3%) through PCA-LDA. Proteomic analysis of SP-EVs revealed 218 proteins in Passed and 238 in Failed samples. Nexin-1 and seminal plasma protein pB1 were upregulated in Passed samples, while LGALS3BP was downregulated. The functional analysis highlighted pathways associated with single fertilization, filament organization, and glutathione metabolism in Passed samples. Integrating NIRS with SP-EV proteomics provides a robust approach to non-invasive assessment of semen quality. These findings suggest that SP-EVs could serve as effective biosensors for rapid semen quality assessment, enabling better boar semen selection and enhancing AI practices in swine breeding. Full article

Extracellular Vesicles (EVs) from seminal plasma have achieved attention due to their potential physiopathological role in male reproductive systems. This study employed a comprehensive proteomic and transcriptomic approach to investigate the composition and molecular signatures of EVs isolated from human seminal plasma. EVs from Normozoospermic (NORMO), OligoAsthenoTeratozoospermic (OAT), and Azoospermic (AZO) subjects were isolated using a modified polymer precipitation-based protocol and characterized for size and morphology. Comprehensive proteomic analysis, using both gel-free and gel-based approaches, revealed distinct protein profiles in each group (p<0.01), highlighting potential molecules and pathways involved in sperm function and fertility. The data are available via ProteomeXchange with identifiers PXD051361 and PXD051390, respectively. Transcriptomic analysis confirmed the trend of a general downregulation of AZO and OAT compared to NORMO shedding light on regulatory mechanisms of sperm development. Bioinformatic tools were applied for functional omics analysis; the integration of proteomic and transcriptomic data provided a comprehensive understanding of the cargo content and regulatory networks present in EVs. This study contributes to elucidating the key role of EVs in the paracrine communication regulating spermatogenesis. A full understanding of these pathways not only suggests potential mechanisms regulating male fertility but also offers new insights into the development of diagnostic tools targeting male reproductive disorders. Full article

Extracellular vesicles (EVs) are gaining recognition for their essential role in enhancing gamete quality and improving outcomes in assisted reproductive technologies. These nanosized particles, released by cells, carry proteins, lipids, and RNAs, facilitating critical cell communication and offering the potential to enhance gamete maturation and improve fertilization rates. Most research on males has concentrated on seminal plasma, a complex fluid produced by the testes and accessory glands vital in modulating sperm fertility potential. The components of seminal plasma significantly affect sperm functionality, embryo survival, and placental development, making this a prominent area of interest in reproductive biology. The EVs within seminal plasma contribute to maintaining sperm membrane stability, enhancing motility, and promoting capacitation, which may influence the female reproductive tract following mating. In females, EVs have been identified in both the follicular and uterine environments, where effective embryo–maternal communication is crucial. The oviduct epithelium supports gamete transport and early embryonic development, with EVs found in oviductal fluid playing a key role in reproductive processes. These EVs support the embryo’s growth in the nutrient-rich uterine environment. These important studies underscore the significant role of EVs in transporting essential molecular compounds to gametes and embryos, leading to an enhanced understanding and potential manipulation of reproductive processes. This review aims to summarize the current research on the benefits of EVs in gamete manipulation and embryo development, highlighting their promising implications for reproductive health. Full article

Background: Although estrogenic compounds promise therapeutic potential in treating various conditions, concerns regarding their endocrine-disrupting effects have been raised. Current methodologies for screening estrogenicity in rodent models are limited to the female-specific uterotrophic bioassay. Studies have reported enlargement of the seminal vesicles in orchiectomized males treated with estrogens. However, identifying estrogenicity strictly through changes in wet weights is uninformative regarding the molecular mechanisms of these agents. Therefore, protein-based biomarkers can complement and improve the sensitivity of weight-based assessments. To this end, we present a discovery-driven proteomic analysis of 17β-estradiol’s effects on the seminal vesicles. Methods: We treated orchidectomized mice with the hormone for five days and used the vehicle-treated group as a control. Seminal vesicles were analyzed by shotgun approach using data-dependent nanoflow liquid chromatography–tandem mass spectrometry and label-free quantification. Proteins found to be differentially expressed between the two groups were processed through a bioinformatics pipeline focusing on pathway analyses and assembly of protein interaction networks. Results: Out of 668 identified proteins that passed rigorous validation criteria, 133 were regulated significantly by 17β-estradiol. Ingenuity Pathway Analysis^® linked them to several hormone-affected pathways, including those associated with immune function such as neutrophil degranulation. The altered protein interaction networks were also related to functions including endocrine disruption, abnormal metabolism, and therapeutic effects. Conclusions: We identified several potential biomarkers for estrogenicity in mouse seminal vesicles, many of them not previously linked with exogenous 17β-estradiol exposure. Full article

Seminal extracellular vesicles (SemEVs) are repositories of biomolecules, including metabolites involved in the regulation of sperm function. The correlation between the metabolite profile of SemEVs and semen parameters, along with their role in regulating sperm function, is an unexplored area. This preliminary study evaluated the metabolomic content of SemEVs. Semen samples were obtained from 18 healthy men, and SemEVs were extracted from seminal plasma using the size exclusion chromatography qEV Gen 2–35 nm column coupled with an automatic fraction collector. The physical characterization of SemEVs was carried out with the ZetaView PMX-430-Z QUATT laser system. EV protein markers were detected using Western blot. In addition, these SemEVs were used for metabolomic profiling and functional bioinformatic analysis. The mean concentration of isolated SemEVs was 1.7 ± 1.1 × 10¹¹/mL of seminal plasma, whereas SemEVs size and zeta potential were 129.5 ± 5.5 nm and −40.03 ± 3.99 mV, respectively. Western blot analysis confirmed the presence of EV specific markers such as CD81, ALIX, and TSG101. A total of 107 metabolites were identified using this untargeted metabolomic approach in SemEVs. Bioinformatics analysis further revealed that metabolites associated with tyrosine metabolism were highly enriched in these SemEVs. Ingenuity Pathway Analysis (IPA) also indicated that these metabolites present in SemEVs were involved in the regulation of the free radical scavenging pathway. Furthermore, our metabolomic results suggest that these SemEV-associated metabolites may play a pivotal role in the maintenance of seminal plasma redox homeostasis. Full article

β-Nerve growth factor (β-NGF) is a protein produced in the reproductive tract of camelids (camels, llamas, and alpacas) that has been identified as the ovulation inducing factor in seminal plasma. β-NGF from seminal plasma deposited into the reproductive tract of the female camelid acts systemically to stimulate the secretion of luteinizing hormone (LH) from the anterior pituitary, which in turn induces follicle maturation and ovulation. The objectives of the present study were to determine if β-NGF is present in the reproductive tract of the stallion and identify the specific site(s) of production. The hypotheses were that β-NGF would be present in the stallion reproductive tract and would primarily be localized in Sertoli cells of the testes and the prostate gland. Immunohistochemistry on paraffin-embedded paraformaldehyde-fixed tissues was performed using a rabbit polyclonal anti-β-NGF antibody on a total of six male equine reproductive tracts, including a one-day old colt, a one-year-old colt, and four adult stallion tracts. Strong immunostaining was observed in the efferent ducts of the testes and the epithelial cells of the prostate, seminal vesicles, bulbourethral glands, and ampullae. Weaker β-NGF staining was noted in Leydig cells, Sertoli cells, and spermatogonia within the testes and in epithelial cells of the epididymis. In conclusion, immunohistochemistry revealed that β-NGF is present in the stallion reproductive tract, and the protein is primarily present in the efferent ducts of the testes and in all accessory sex glands. Full article

Accessory sex glands are recognized as targets of human disease and may have roles in reproductive success in livestock. The current experiments evaluated the influences of endogenous steroids on the development of porcine accessory sex glands, primarily in the neonatal period. When the aromatase inhibitor, letrozole, was used to inhibit the production of endogenous estrogens in the postnatal interval, growth of the seminal vesicles, prostate, and bulbourethral glands was stimulated. The weights of seminal vesicles, prostate, and bulbourethral glands approximately doubled at 6.5 weeks of age when the reduction in endogenous estrogens began at 1 week of age (p < 0.01). However, by 20 and 40 weeks of age, the weights of accessory sex glands were similar between the letrozole-treated boars and the vehicle-treated littermates indicating the growth stimulation was a transient effect when the treatment interval was short. The presence of both classical nuclear estrogen receptors and the G protein-coupled estrogen receptor in neonatal accessory sex glands indicated multiple signaling pathways might mediate the growth inhibition by endogenous estrogens. The absence of a detectable response when the classical estrogen receptors were blocked with fulvestrant (or when the androgen receptor was blocked with flutamide) suggests that endogenous estrogens act through the G protein-coupled estrogen receptor to inhibit the development of accessory sex glands during this neonatal to early juvenile interval. Full article

Background: Ubiquitin-specific protease 11 (USP11), one of the principal phosphatase and tensin homolog (PTEN) deubiquitinases, can reserve PTEN polyubiquitination to maintain PTEN protein integrity and inhibit PI3K/AKT pathway activation. The aim of the current study was to investigate the associations between immunohistochemical USP11 staining intensities and prognostic indicators in individuals with prostate cancer. Methods: Tissue microarrays (TMAs) were performed for human prostate cancer and normal tissue (control) samples. Data on patient’s age, Gleason score, plasma prostate-specific antigen (PSA) titer, disease stage, and presence of seminal vesicles, lymph nodes, and surgical margin involvement were collected. A pathologist who was blinded to the clinical outcome data scored the TMA for USP11 staining intensity as either positive or negative. Results: Cancerous tissues exhibited lower USP11 staining intensity, whereas the neighboring benign peri-tumoral tissues showed higher USP11 staining intensity. The degree of USP11 staining intensity was lower in patients with a higher PSA titer, higher Gleason score, or more advanced disease stage. Patients who showed positive USP11 staining were more likely to have more optimal clinical and biochemical recurrence-free survival statistics. Conclusions: USP11 staining intensity in patients with prostate cancer is negatively associated with several prognostic factors such as an elevated PSA titer and a high Gleason score. It also reflects both biochemical and clinical recurrence-free survival in such patients. Thus, USP11 staining is a valuable prognostic factor in patients with prostate cancer. Full article