Sperm Activation Research Articles

Association of NOX5 Expression with Sperm Activity and Motility in Pathospermic Infertile Men

Background: The newest NOX isoform, NOX5, has been found in mammalian spermatozoa. Many physiological and pathological situations in spermatozoa are mediated by reactive oxygen species (ROS). NOX5 is the main source of ROS in spermatozoa. Our purpose was to investigate the changes in NOX5 expression and the effect of NOX5 expression on sperm motility, chromatin integrity, and oxidative status in oligoasthenozoospermic compared to normozoospermic men. Methods: Semen samples were collected from 30 normozoospermic (NS) and 30 oligoasthenozoospermic (OAS) men. NOX5 protein expression in sperm samples was evaluated by immunohistochemistry and western blot. Oxidative stress status was evaluated by total antioxidant capacity (TAC), total oxidant capacity (TOC), and oxidative stress index (OSI) parameters. Chromatin integrity in spermatozoa was evaluated by toluidine blue staining. Results: NOX5 expression levels were significantly higher in OAS group than in NS group (p<0.001). In addition, chromatin integrity was significantly higher in the OAS group in comparison to NS group (p<0.001). TAC levels were higher in the NS group, but OSI and TOC levels were significantly higher in OAS group (p<0.001). It was found that NOX5 protein expression was positively correlated with oxidative stress and chromatin integrity and negatively correlated with motility (p<0.01). Conclusion: These results suggest that overexpression of NOX5 may be the source of excessive ROS production and oxidative stress injuries in oligoasthenozoospermic men. Considering that NOX5 expression is positively correlated with oxidative stress and chromatin integrity but negatively correlated with motility, it can be considered a biomarker to be used in assisted reproductive procedures.

The Impact of NADPH Oxidase 5 Activity and Reactive Oxygen Species on Ca-pacitated Human Sperm

Background: Progesterone (P4) activates sperm calcium channels (CatSper), allowing calcium to enter the cell, which activates NADPH Oxidase-5 (NOX5) and produces reactive oxygen species (ROS). While calcium and ROS are essential for sperm capacitation, the role of NOX5 in capacitated sperm is unclear. This study investigated NOX5 activity in capacitated human sperm. Methods: Forty semen samples from fertile men were processed, with motile sperm separated and divided into nine groups: control (Ham's F-10), solvent (DMSO), progesterone, diphenyleneiodonium chloride (DPI, NOX5 inhibitor), phorbol-12-my-ristate 13-acetate (PMA, NOX5 activator), P4+DPI, P4+PMA, Trolox, and P4+ Trolox. Sperm kinematics, membrane integrity, survival rate, and ROS production was evaluated. Data were analyzed using ANOVA and Kruskal-Wallis tests, p£ 0.05 considered statistically significant. Results: Progressive motility significantly decreased with DPI (56.2±2.1%) and PMA (56.5±2.1%), both alone and combined with progesterone (58.0±2.0% and 57.4±2.2%), compared to the progesterone group (66.0±1.9%). No significant change was observed in the Trolox groups. Progesterone, alone or combined with DPI, PMA, and Trolox, significantly reduced sperm linearity from 0.6±0.01 to 0.5± 0.01%. Straight-line velocity decreased in P4+PMA and P4+Trolox groups (88.2± 4.4 and 89.7±3.9 μm/s) compared to the control group (105.0±5.5 μm/s). Trolox reduced ROS content, while other treatments had no effect on ROS levels. Conclusion: NOX5 does not play a prominent role in capacitated sperm. The negative effects of PMA and DPI on sperm motility appear independent of their actions on NOX5 and ROS production. Trolox did not affect sperm motility and survival, indicating that capacitated sperm require little or no ROS.

LC-MS metabolomics uncovers potential biomarkers of semen cryo-injury in goats.

Semen cryopreservation acts a crucial role in enhancing breed improvement and conserving genetic resources. However, it often leads to decreased sperm activity and reduced pregnancy rates. Despite significant advancements in semen freezing techniques for goats, the precise factors and mechanisms causing cryo-injury remain unclear. In this study, we examined the motility characteristics of fresh semen versus frozen-thawed semen and investigated changes in the metabolite profiles of seminal plasma using liquid chromatograph-mass spectrometry (LC-MS). A total of 364 differentially expressed metabolites (DEMs) were identified between fresh and frozen-thawed semen samples. Among these, 185 metabolites were significantly up-regulated, while 179 were down-regulated (p<0.05). The majority of these DEMs belonged to lipids and lipid-like molecules, as well as organic acids and derivatives. The Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that these DEMs were primarily involved in pathways related to amino acid synthesis and metabolism. Additionally, metabolite set enrichment analysis (MSEA) underscored the critical role of amino acid synthesis and metabolic pathways in semen cryopreservation. Specific metabolites such as alanine, proline, phenylalanine, tryptophan, tyrosine, adenosine, citric acid, flavin adenine dinucleotide (FAD), and choline emerged as potential biomarkers for sperm cryo-injury in goats. These findings provide valuable insights into enhancing the quality of semen cryopreservation in goats, contributing to improved breeding and genetic resource conservation efforts.

Cryoprotective Potential of Theobromine in the Improvement of the Post-Thaw Quality of Bovine Spermatozoa.

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [CtrlC]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (CtrlN). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity (p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to CtrlC. All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa (p < 0.001) than CtrlC. Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR (p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 (p < 0.01), while all TBR doses stabilized the levels of PKC (p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

Correlation of Seminal Plasma EMMPRIN Levels with Sperm Quality and the Efficacy of Activation Techniques

Background: Infertility is a major global health challenge, impacting 15% to 20% of couples globally. Male infertility, which is typically associated with issues such as low sperm motility, has increased during the last two decades. Male fertility relies heavily on the development of extracellular matrix metalloproteinase inducer (EMMPRIN), a glycosylated transmembrane protein involved in sperm function and embryo implantation. Understanding how sperm preparation techniques affect sperm quality and EMMPRIN expression is crucial for achieving optimal results in assisted reproductive technologies (ART). Objective: To investigate EMMPRIN expression in human sperm and compare the impact of two in vitro sperm preparation procedures, “direct swim-up (DSU) and pellet swim-up (PSU), on various sperm characteristics. To determine the optimum technique for improving sperm quality in ART. Materials and Methods: Masturbation was employed to collect sperm samples from 44 participants (aged 21 to 45). Following liquefaction, samples were evaluated macroscopically and microscopically in accordance with WHO 2021 guidelines. The samples were separated into three aliquots for ELISA assessment of EMMPRIN levels and sperm processing with DSU and PSU techniques. Sperm chromatin immaturity (SCI) was assessed using Aniline Blue staining. Statistical investigations were carried out to determine the correlations between EMMPRIN levels, semen parameters, and the impacts of the two in vitro sperm preparation techniques. Results: Sperm concentration significantly decreased from control (40.25 ± 2.30) to DSU (15.51 ± 1.74) and PSU (9.99 ± 1.16) conditions. However, motility, morphology, and SCI significantly improved, particularly with PSU, which showed a greater reduction in SCI % compared to DSU, indicating its effectiveness in selecting higher-quality sperm. There were no significant associations detected between EMMPRIN levels with semen metrics, although PSU exhibited a slightly higher association with improved sperm parameters. Conclusion: Seminal plasma EMMPRIN levels do not appear to directly influence semen parameters or sperm chromatin maturity. However, the choice of sperm activation technique, particularly PSU, significantly impacts sperm quality. More research is needed to better understand the importance of EMMPRIN in ART outcomes and its potential as a sperm quality indicator.

DNA methylation correlates with transcriptional noise in response to elevated pCO2 in the eastern oyster (Crassostrea virginica).

Ocean acidification significantly affects marine calcifiers like oysters, warranting the study of molecular mechanisms like DNA methylation that contribute to adaptive plasticity in response to environmental change. However, a consensus has not been reached on the extent to which methylation modules gene expression, and in turn plasticity, in marine invertebrates. In this study, we investigated the impact of pCO2 on gene expression and DNA methylation in the eastern oyster, Crassostrea virginica. After a 30-day exposure to control (572 ppm) or elevated pCO2 (2827 ppm), whole-genome bisulfite sequencing (WGBS) and RNA-seq data were generated from adult female gonad tissue and male sperm samples. Although differentially methylated loci (DMLs) were identified in females (89) and males (2916), there were no differentially expressed genes and only one differentially expressed transcript in females. However, gene body methylation impacted other forms of gene activity in sperm, such as the maximum number of transcripts expressed per gene and changes in the predominant transcript expressed. Elevated pCO2 exposure increased gene expression variability (transcriptional noise) in males but decreased noise in females, suggesting a sex-specific role of methylation in gene expression regulation. Functional annotation of genes with changes in transcript-level expression or containing DMLs revealed several enriched biological processes potentially involved in elevated pCO2 response, including apoptotic pathways and signal transduction, as well as reproductive functions. Taken together, these results suggest that DNA methylation may regulate gene expression variability to maintain homeostasis in elevated pCO2 conditions and could play a key role in environmental resilience in marine invertebrates.

High temperature induces motility of zebrafish sperm after short-term storage

Short-term storage on ice of zebrafish (Danio rerio) sperm has been successfully applied in diverse basic experimental research. The optimal and economically efficient short-term storage method to date, however, can only maintain the fertilization ability of the sperm for a few hours. In this study, we aimed to improve zebrafish sperm quality after short-term storage and to test whether this affects the nucleotide cargo of spermatozoa. Zebrafish sperm was diluted with E400 extender (130 mM KCl, 50 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM D-(+)-Glucose and 30 mM HEPES-KOH, pH 7.9, 400 mOsmol/kg) at a dilution ratio 1:20 (v/v) in tubes (500 μL) and stored for 24 h at 0–2 °C under aerobic conditions. Elevating the temperature of sperm suspension at 28 °C for 20 min significantly enhanced sperm motility during activation in both distilled water and Perchec solution with 0.25 % Pluronic F-127. Despite the observed decline in sperm motility during short-term storage, Perchec solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0, 160 mOsmol/kg) enhanced activation of the aged sperm compared to activation of distilled water. Following 24 h of sperm storage, higher fertilization, and hatching rates were observed when sperm suspension was incubated at 28 °C for 20 min and activated in Perchec solution at a ratio of approximately 6120 spermatozoa per egg, compared to activation in hatchery water. A tentative RNA-seq study identified a set of 2180 differentially expressed genes after 24 h of sperm storage, significantly enriched in the glycosaminoglycan degradation and galactose metabolism pathways. The validity was discussed due to the limited mRNA content in spermatozoa. No alterations were identified in the DNA methylation level of 24 h stored sperm and neither differentially methylated CpGs (DMCs) nor differentially methylated regions (DMRs) were detected between fresh and aged sperm. Based on these phenotypic and molecular investigations and the successful implementation of high-temperature incubation prior to sperm activation in artificial reproduction, we propose that high-temperature incubation following short-term sperm storage at low temperatures is a safe and significant approach in zebrafish and holds potential benefits for other economically important warm-water species in aquaculture.

Effect of different concentrations of inulin on ram sperm quality during cryopreservation

BACKGROUND: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation. OBJECTIVES: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing. MATERIALS AND METHODS: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196°C. RESULTS: The total motility, viability, and DNA integrity were significantly improved after freezet-hawing with 28 mM inulin, compared to other treatment groups (P&lt;0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p&lt;0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P&lt;0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P&lt;0.05). CONCLUSION: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm.

Integrated sperm movement, proteomics, and phosphoproteomics elucidate the roles of osmolality, ions, and key proteins in sperm activation of Nile tilapia (Oreochromis niloticus)

Nile tilapia (Oreochromis niloticus) is an important aquaculture fish around the world and has a wide range of tolerance to salinity, providing great economic value in aquaculture industry and showing an excellent ecological adaptation. However, the best sperm activation condition and sperm activation mechanism of this euryhaline fish remains unclear. In this study, we applied the sperm movement analysis, proteomics, and phosphoproteomics to explore the effects of osmolality and ions and to reveal sperm activation mechanism of Nile tilapia. Our results showed that low osmolality contributed to sperm activation compared with high osmolality. Adding Na+ and Ca2+ enhanced sperm motility after sperm activation. Under 100 mOsm·kg−1, adding 44 mM Na+ and 4 mM Ca2+ was a successful protocol for sperm activation. Proteomics and phosphoproteomics analyses identified 677 differentially expressed proteins and 245 differentially expressed phosphorylated proteins, respectively, among which 21 proteins and 11 phosphorylated proteins as key molecules were involved in Ca2+ mediated signal transduction, glucose metabolism, lipid metabolism, and apoptotic cell death. Key protein ORAI1 promoted extracellular Ca2+ influx to activate Ca2+ mediated signal transduction. Converting glycogenolysis and glycolysis to pyruvate metabolism and fatty acid β-oxidation was a critical energy metabolic adaptation mechanism. The Hsp90 was a critical sperm activation biomarker in loosing of sperm motility and apoptosis. A putative proteomic and phosphoproteomic response network was constructed to reveal the sperm activation mechanism of Nile tilapia. Our study provides a new insight into sperm activation mechanism of this euryhaline fish and facilitates future application of best activation protocol to the fertilization and breeding of Nile tilapia.

Novel combination with maca improves sperm parameters in vitro of asthenozoospermic men.

Infertility is an inability to conceive after a reasonable period of time (12 months) without the use of contraception or due to a person's incapacity to procreate, whether independently or with a spouse. Problems with the production and maturation of sperm are the most common causes of male infertility additionally; the motility is the major functional character that determines the fertilizing ability of spermatozoa. Therefore the goal of this study is to get better certain sperm function parameters in vitro of asthenozoospermic patient. The World Health Organization (WHO) and many studies considered the infertility as a disease and so many couples complaining from unsuccessful assisted reproductive technologies (ART) procedures to overcome their problem. The goal of this study is to improve certain sperm function feature in vitro of asthenozoospermic semen patients by using combination of motility inducing namely; Maca, L-carnitine and Pentoxifylline that enhance the medium to improve certain sperm characters that might be utilized for ART centers. Semen aliquots were collected from ninety patients with asthenozoospermia who participated in present study, the volume of semen samples with normal ejaculate when was ranged between 1.4-6ml and can be measured by using a measure pipette or conical graduated tube; Inclusion criteria was Asthenozoospermia, oligozoospermia and teratozoospermia men, Infertile idiopathic men also, fertile normozoospermic men. While Exclusion criteria was Azoospermic men, Alcoholic, Patients under treatment with antibiotics and men with Varicocele.The samples split into two equal groups at random. Using Ham's F12 medium, one portion served as the control group, and the other was the treatment group, which was mixing by combining the following ingredients, Maca powder extracts (Lepidium meyenii) (M) 1 mg/ml, 0.5 mg/ml of L-Carnitine (LC), and 10 mg/ml of Pentoxifylline (PTX). The data were analyzed using Statistical Package for Social Sciences (SPSS) version 23.0. The descriptive statistics including frequency, range, mean and standard error, Data from treated and control groups were expressed as mean ± SEM and to compare value between experimental and control groups using Students t-test. Layering approach is used to investigate sperm parameters before and after in vitro activation. The information showed a very large (p< 0.001) increase in active sperm motility grade A, percentage of progressive motility with significant increase in morphologically normal sperm (MNS) with decreased in DNA fragmentation index after activation by layering technique with novel combination medium compared to Hams F12 medium and before activation. The present work stated that novel combination medium (LC, maca and PXT) have potential effects to improve sperm characters in male infertility factors and suggested to be used for sperm preparation and activation in ART programs.

Effects of Melatonin on Spermatozoa Activity and Associated Mechanisms in Heat-Stressed Rats

Effects of Melatonin on Spermatozoa Activity and Associated Mechanisms in Heat-Stressed Rats

Comparative Investigation of the Mechanisms of Calcium Response in Human and Murine Spermatozoa

Calcium signaling is a principal method of signal transduction in cells of non-excitable tissues. In both mouse and human sperm, it can be induced in response to progesterone, manifesting as oscillations or single peaks and followed by the acrosomal reaction. However, the molecular mechanisms of progesterone activation may vary between species. In this study, we aim to compare the calcium signaling mechanisms in human and mouse spermatozoa. We investigated the calcium response in mouse sperm activated by progesterone. We employed spectrofluorometry to quantify the rise in calcium concentration in response to progesterone in Fura-2 loaded mouse sperm cells in suspension. Our experiments demonstrated that mouse sperm cells respond to 50 μM progesterone with a peak 120 ± 35 s wide and 0.8 ± 0.3 μM high. Based on literature data, a scheme for the induction of calcium signaling was constructed, suggesting an intermediate stage with the synthesis of a certain prostanoid (possibly PGE2) and activation of mouse sperm by this prostanoid through a G-protein-coupled receptor. Based on the obtained reaction scheme, two computational models were developed: a point model and a three-dimensional model. As with human sperm, the point model provided only a qualitative description of calcium responses, whereas the three-dimensional model produced the shape of the calcium peak and the frequency of calcium oscillations in response to progesterone that were similar to the experimentally obtained values. Using in silico analysis, it was shown that in mouse sperm, the spatial distribution of signaling enzymes regulates the type and form of the calcium response. We conclude that the presence of time delays due to the diffusion and spatial distribution of calcium signaling enzymes regulates the calcium response in both human and mouse sperm.

ANALYSIS OF VARIANCE SPERMOGRAM INDICATORS OF MEN DIFFERENT AGE GROUPS WITH ASTENOZOOSPERMIA, TERATOZOOSPERMIA AND AZOOSPERMIA

In recent decades, a decline in male fertility due to deterioration in sperm quality hasbeen noted around the world. This is probably explained by the tendency to increase the diseasesof the male genital organs and, as a result, the increase in the percentage of male infertility.In addition, there is a deterioration of the quantitative and qualitative indicators of spermogramsin practically healthy men. The average number of sperm in the ejaculate of a healthy man hashalved over the past 50 years, and the average volume of ejaculate has decreased by one third. Itis known that the multicomponent composition of the internal male genital organs is in constantrestructuring due to age changes, functional activity and the influence of various factors. That iswhy it is important to take into account both physiological and age-related changes in a man'sability to conceive.Diagnosis of male infertility includes clinical research methods and methods of laboratory-instrumental examination. Among the latter, the most important for finding out the functionalstate of the gonads and the fertilizing ability of sperm is the study of ejaculate. The object ofour research was spermograms obtained during the examination of patients at the "AlternativeClinic" reproductive medicine clinic.The purpose of the work was using biometric analysis to the different indicators of menspermograms of younger, middle and older age groups with normozoospermia (N), asthenozoospermia(AZS), teratozoospermia (T) and azoospermia (A). The task of the research wasto analyze the main indicators of spermograms of men of different age groups in normal andpathological conditions, to conduct a one-factor and two-factor variance analysis of the influenceof the studied diseases and age factor.After analyzing the main indicators of spermograms of men of younger, middle andolder age groups with asthenozoospermia, azoospermia, and teratozoospermia, it was establishedthat the indicators that undergo the greatest deviations in the studied diseases are themobility of spermatozoa according to categories A and B, the morphology of spermatozoa, theFarris fertility index, and the activity of spermatozoa.After conducting a one-factor variance analysis, we established that the share of theinfluence of the studied diseases on the overall variability of such indicators of spermograms asthe mobility of spermatozoa according to category A and B and the morphology of spermatozoain the spermograms of men of the studied age groups is within 63–98% of the total contribution(younger age group), 60–96% (middle age group) and 75–96% (older age group). The shareof influence of unaccounted factors is within 2–40% of the total contribution. The share of theinfluence of the studied diseases in the overall variability of such indicators as the Farris index and sperm activity in the spermograms of men of all studied groups is significantly reducedand is within 22–44% of the total contribution. Instead, the influence of unaccounted factors(56–78% of the total contribution) is growing significantly. This may indicate the presence ofconcomitant diseases and other pathologies in the men who took part in the research.After conducting a two-factor variance analysis, we established that the shares of theinfluence of the studied diseases on the overall variability of such indicators of spermogramsas motility of spermatozoa according to category A and B, morphology of spermatozoa, Farrisfertility index and activity of spermatozoa are decisive and are within the range of 90.7–99.9%of the total contribution, the share of the influence of the age factor on the variability of spermogramindicators of men of different age groups is insignificant.

P-025 Mitochondrial activity in sperm is associated to blastocyst development: a double-donation study

Abstract Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P&amp;gt;0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

P-097 Studying epididymal sperm using cryo-electron microscopy

Abstract Study question What are the structural changes in epididymal sperm leading to gain of motility? Summary answer We resolved the contents of the cytoplasmic droplet in immature sperm. We further found that the outer dynein arms are in a pre-power stroke conformation. What is known already Cells exiting the testis are morphologically complete but incapable of fertilization. They first need to mature in the epididymis, where sperm gain progressive motility. Intriguingly, during this epididymal maturation the cytoplasmic droplet, a cytoplasmic remnant from spermiogenesis, migrates distally from the connecting piece to the principal piece. At the heart of the sperm’s flagellum is the axoneme, the molecular motor of the cell. It is a microtubule-based structure that houses protein complexes with different functions. For instance, it has radial spokes for signaling and rigidity. It also houses dyneins, motor proteins attached to neighboring doublets that ultimately generate flagellar beating. Study design, size, duration Bovine testes were acquired from a slaughterhouse, and sperm were isolated from different regions of the epididymis. After a washing step, these cells were directly used for imaging. Participants/materials, setting, methods Cells were prepared for imaging by rapid freezing on cryo-Electron Microscopy (cryo-EM) grids. Frozen cells were either imaged directly or first thinned by Focused Ion Beam (FIB) – milling. We applied the image analysis approaches subtomogram averaging and single particle analysis. Main results and the role of chance We resolved the structure of the 96-nm repeat of the axonemal doublet from immature sperm and showed a complete axoneme, containing radial spokes and different sets of dynein arms. Focusing our refinement on a specific set of dyneins, the outer dynein arms, revealed that they are in the pre-power stroke state. In this primed state the dyneins have ATP bound and are active. Interestingly, these cells display no movement. This could indicate that a different blocking mechanism is present in the axoneme. Furthermore, we used 3D cryo-EM to elucidate the contents of the cytoplasmic droplet. We showed that it contains many vesicles, some display a shape that is reminiscent of an autophagosome. We also find vesicles displaying a double membrane with a highly repetitive density connecting in between. This suggests that sperm cells bring along auxiliary proteins and molecules in the cytoplasmic droplet that may be required for proper maturation. Altogether we show that cryo-EM can be used to study immature sperm cells in detail, with a focus on structures important in the gain of movement. Limitations, reasons for caution Cryo-EM imaging requires frozen cells and uses electrons as imaging source, therefore we lack information on cell state (e.g. viability/motility) or protein identity. By complementing our visual data with other techniques such as CASA and mass spectrometry, we aim to get a full picture. Wider implications of the findings Elucidating protein conformation or the structural mechanism behind sperm activation could open doors into developing new therapeutics for diseases on one hand. On the other hand, understanding how sperm motility arises (and how to potentially put it to a halt) could also lay ground for developing male contraception. Trial registration number not applicable

Efficacy of GnRH Pulses in Hypogonadism Secondary to Primary Empty Sella: Case Report.

This study aims to assess the effectiveness of pulsed gonadotropin-releasing hormone (GnRH) micropump replacement therapy in the treatment of hypogonadotropic hypogonadism (HH) caused by primary empty sella (PES).The efficacy of pulsed GnRH replacement therapy using the micropump was evaluated in a middle-aged male patient with HH who had experienced the loss of his only child. Relevant literature was also consulted to compare the differences between pulse GnRH treatment and conventional treatment in terms of the development of secondary sexual characteristics, sex hormone levels, sperm production rate, and sperm activity rate in male patient with HH.In this report, a 45-year-old male diagnosed with HH and PES presented with fatigue and decreased libido. The main characteristics included decreased follicle stimulating hormone (FSH) levels of 0.03 mIU/mL, luteinizing hormone (LH) levels of 0.02 mIU/mL, and testosterone (T) levels of 0.72 nmol/L. Magnetic resonance imaging (MRI) revealed an empty sella. Semen analysis showed a small number of normal sperm with reduced motility. During treatment with the micropump pulse GnRH, the patient experienced no side effects and showed improvements in fatigue, reduced libido, sexual urge, anxiety, and feelings of inferiority. LH, FSH, and T levels returned to normal, while sperm activity rate increased to 79.9%. Ultimately, the patient's spouse achieved a natural pregnancy.Pulsed gonadotropin delivery using the micropump demonstrates good efficacy and tolerability, and aligns more closely with the physiological rhythm of GnRH secretion in the human body.

Impact of seasonality on the reproductive physiology of diploid and tetraploid eastern oyster, Crassostrea virginica

Aquaculture production of triploid Eastern oyster, Crassostrea virginica, in the Gulf of Mexico (GoM) is reliant on naturally conditioned diploid females and tetraploid males. However, limited knowledge is available on their reproductive biology, including the gametogenic cycle. The aim was to quantify monthly changes in gametogenesis and gamete quality for triploid production in Eastern oyster in the GoM from June 2022 to July 2023. Monthly changes in temperature and salinity were recorded. Shell morphometrics and sex were determined. Sperm density, sperm activity, Spermatogenic Maturity Index (SMI), oocyte diameter, Oogenesis Maturity Index (OMI), and gonad coverage area were quantified. Mean monthly temperature ranged from 15.1 to 31.2 °C and salinity ranged from 12.5 to 27.6 PSU. Shell morphometrics increased over the annual cycle. When gametes were detectable, 48–80% of tetraploids and 45–70% of diploids were males. Hermaphrodites were identified in both ploidies in June; an additional tetraploid hermaphrodite was identified in March. Sperm density exhibited monthly fluctuations with high densities reported in June–July, September, and April–May. Sperm were motile when present, with high motility in June–July. High sperm velocity was also recorded in June–July and March–May. Tetraploids exhibited high SMI from June–September and April–May with increased variability in December–January. Mature sperm cells were predominant in tetraploids in June–July. Gonadal coverage was high from June–September and March–May. OMI was highest from June–September and March–May, while variability was high from August–March. Large oocyte diameters and gonad coverage areas were observed in June–July, September, and April–May. Together, these findings provide novel insights into reproductive dynamics for triploid production of Eastern oyster in GoM.

Ultrasound-assisted deep eutectic solvents extraction of polysaccharides from Loquat leaf: Process optimization and bioactivity study

Loquat leaves are the by-product of loquat fruit production. Polysaccharides are one of the main active ingredients in loquat leaves. In this study, polysaccharides were extracted from loquat leaves by ultrasonic-assisted deep eutectic solvents (DESs) extraction method. Further, the extracted crude loquat leaf polysaccharides (CLLP) were purified and separated via S-8 resin and DEAE-52 cellulose column chromatography, respectively. Additionally, the effects of polysaccharides on activity of sperm in boar semen preserved in medium at 17 °C, were evaluated preliminarily. DES, composed of choline chloride/ethylene glycol (1:6, molar ratio), was proved to be the suitable solvent for LLP extraction. The optimized extraction conditions were water content 44 %, liquid-solid ratio 1:29 (g/g), extraction temperature 61 °C and extraction time 98 min. Under these conditions, the LLP yield was 57.82 ± 1.50 mg/g. A homogeneous polysaccharide (LLP1-2, Mw: 2.17 × 104 Da) was isolated from CLLP. Its total sugar, uronic acid and protein contents were 76.31 ± 1.25 %, 14.19 ± 0.67 % and 3.28 ± 0.42 %, respectively. Further, 800 μg/mL LLP1-2 could effectively enhance the antioxidant activity of sperm. This study laid a foundation for DESs and column chromatography in the field of polysaccharide extraction and separation, proving that LLP can be used as a natural antioxidant for sperm preservation.

Regulation of sperm motility by exosomes of ovarian fluid during female sperm storage in black rockfish (Sebastes schlegelii)

Sperm storage within the female reproductive tract is a widespread phenomenon among vertebrate species, including various viviparous teleosts. The black rockfish (Sebastes schlegelii) serves as a typical example, wherein sperm remains stored inside the ovary for extended periods, spanning over four months from entry until fertilization. While previous investigations have explored the occurrence of female sperm storage and its adaptive advantages, the precise mechanisms governing sperm storage in the ovary and the maintenance of sperm activity remain incompletely understood. Recently, extracellular vesicles or exosomes have been identified as important components of the oviductal or uterine fluid, but their interactions with sperm are not fully understood. Our study represented the isolation and identification of ovarian fluid exosomes (Ov-exos) from the black rockfish, while also affirming their role in sustaining sperm activity. Based on the transcriptome and proteome of the ovaries, it was found that the levels of gene transcription and protein translation, which related to exosomes, were upregulated compared to pre-mating levels, such as CD81, CD63, CD9, ANXA1, VPS25, and RAB8A. Furthermore, our study corroborated these findings through qRT-PCR analysis. Immunohistochemical localization of CD81 suggested that exosomes were secreted at the sperm storage site within the ovary. The ultrastructure of ovaries, particularly with exosomes, was examined by transmission electron microscopy (TEM). It was further found that epithelial cells and follicular layer cells secreted exosomes, which bound to the mid-piece of sperm stored in the ovaries. To validate our findings, experiments involving the co-incubation of sperm with Ov-exos were conducted. This analysis demonstrated that exosomes could bind with sperm and transfer their cargo, significantly enhancing sperm viability and motility. Notably, this study helps to reveal the complexity of sperm storage mechanisms and their adaptive implications in teleost reproduction.