Maintain Sperm Function Research Articles

Extracellular vesicles in porcine seminal plasma maintain sperm function by reducing Lyso-PC

Seminal plasma (SP) exosomes are membrane vesicles with 30−120 nm diameter, which have been shown to regulate the physiological processes in sperm cells. However, the mechanism through which SP extracellular vesicles (EVs) maintain sperm function remains unclear. To explore the mechanism of EVs in sperm preservation, different EV concentrations (EV-0, EV-1, EV-4, EV-8, EV-16) were added to semen liquid preservation solution. Sperm motility was detected via computer-assisted sperm analysis systems, plasma membrane integrity was assessed using SYBR-14 and propidium iodide staining, and total antioxidant capacity and lipid oxidation levels were evaluated after 0, 2, 4, 6 days of liquid-storage at 17 °C. Differences in sperm metabolites between the EV-0 and EV-8 groups stored for 4 days, were analyzed using untargeted metabolomics [liquid chromatography with tandem mass spectrometry (LC-MS/MS)]. Moreover, mitochondrial reactive oxygen species (mtROS) levels were assessed when sperm incubated with EV-0 and EV-8 during 0, 2, 4, 6 days, and embryonic development were assessed when sperm incubated with EV-0 and EV-8 stored for 4 days at 17 °C. Our data showed that compared to EV-0, sperm motility and plasma membrane integrity, antioxidant capacity, lipid oxidation levels and cleavage rates were significantly increased in the EV-8 group (P < 0.05), lysophosphatidylcholine (Lyso-PC) metabolisma and mtROS levels were significantly reduced in the EV-8 group (P < 0.05). In conclusion, SP EVs inhibit the premature increase in Lyso-PC, exert antioxidant effects on mtROS, and preserve sperm's ability to fuse with oocytes.

Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

Deactivation of the JNK Pathway by GSTP1 Is Essential to Maintain Sperm Functionality.

Fifty percent of male subfertility diagnosis is idiopathic and is usually associated with genetic abnormalities or protein dysfunction, which are not detectable through the conventional spermiogram. Glutathione S-transferases (GSTs) are antioxidant enzymes essential for preserving sperm function and maintaining fertilizing ability. However, while the role of GSTP1 in cell signaling regulation via the inhibition of c-Jun N-terminal kinases (JNK) has been enlightened in somatic cells, it has never been investigated in mammalian spermatozoa. In this regard, a comprehensive approach through immunoblotting, immunofluorescence, computer-assisted sperm assessment (CASA), and flow cytometry analysis was used to characterize the molecular role of the GSTP1–JNK heterocomplex in sperm physiology, using the pig as a model. Immunological assessments confirmed the presence and localization of GSTP1 in sperm cells. The pharmacological dissociation of the GSTP1–JNK heterocomplex resulted in the activation of JNK, which led to a significant decrease in sperm viability, motility, mitochondrial activity, and plasma membrane stability, as well as to an increase of intracellular superoxides. No effects in intracellular calcium levels and acrosome membrane integrity were observed. In conclusion, the present work has demonstrated, for the first time, the essential role of GSTP1 in deactivating JNK, which is crucial to maintain sperm function and has also set the grounds to understand the relevance of the GSTP1–JNK heterocomplex for the regulation of mammalian sperm physiology.

Palmitoylated GLB1L4 transfers via exosomes to maintain sperm function in rat epididymis.

Epididymal specific proteins play a crucial role in sperm maturation. Some of the post-translational modified proteins are transported from the caput to the cauda of the epididymis through exosomes which regulate the function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expressed specifically in the caput epididymis, localizes on the sperm; however, the regulatory ways in which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce the fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at the 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes.

Post-ejaculation thermal stress causes changes to the RNA profile of sperm in an external fertilizer.

Sperm cells experience considerable post-ejaculation environmental variation. However, little is known about whether this affects their molecular composition, probably owing to the assumption that sperm are transcriptionally quiescent. Nevertheless, recent evidence shows sperm have distinct RNA profiles that affect fertilization and embryo viability. Moreover, RNAs are expected to be highly sensitive to extracellular changes. One such group of RNAs are heat shock protein (hsp) transcripts, which function in stress responses and are enriched in sperm. Here, we exploit the experimental tractability of the mussel Mytilus galloprovincialis by exposing paired samples of ejaculated sperm to ambient (19°C) and increased (25°C) temperatures, then measure (i) sperm motility phenotypes, and (ii) messenger RNA (mRNA) levels of two target genes (hsp70 and hsp90) and several putative reference genes. We find no phenotypic changes in motility, but reduced mRNA levels for hsp90 and the putative reference gene gapdh at 25°C. This could reflect either decay of specific RNAs, or changes in translation and degradation rates of transcripts to maintain sperm function under stress. These findings represent, to our knowledge, the first evidence for changes in sperm RNA profiles owing to post-ejaculation environments, and suggest that sperm may be more vulnerable to stress from rising temperatures than currently thought.

Human sperm vitrification: A scientific report.

The sperm vitrification developed by this group is based on the ultrarapid freezing of a vitrification solution composed of a non-permeable cryoprotectant (saccharides and protein), in which previously selected spermatozoa are resuspended, free of seminal plasma, and then plunged directly into liquid nitrogen. Compared to traditional sperm freezing, vitrification does not cause chemical or physical damage to the intracellular structures and reduces the damage to the plasma membrane because no ice crystals form, thus preserving motility and DNA integrity. This manuscript is a review of the vitrification methodology developed by the authors' research group, including studies showing the application in human reproduction therapy. The authors perform a review of the work initiated more than a decade ago by this research group, on the implementation of sperm vitrification, a more effective technique for cryopreservation of human spermatozoa, discussing the results obtained by other authors and the projection of this technique. The vitrification technique has been developed in selected spermatozoa free of seminal plasma supplemented with saccharides such as sucrose, trehalose, and dextran, together with albumin, providing a high motility rate and protective structures of the cytoskeleton. In patients, it can be used to preserve their fertility for oncological reasons, genetics, inflammatory diseases, or reproductive medicine techniques. The possibility that vitrified spermatozoa can be preserved at temperatures of -80°C can simplify sample storage, optimizing the space and time as well as operator safety. Vitrification techniques have demonstrated the preservation of selected spermatozoa without seminal plasma and with non-permeable cryoprotectants and protein. Currently, it is one of the most effective ways to maintain sperm function and has been used in in vitro fertilization or intrauterine insemination in humans, achieving healthy live births.

Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination.

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen's genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

Cryopreservation of cynomolgus macaque (Macaca fascicularis) sperm with glycerol and ethylene glycol, and its effect on sperm-specific ion channels — CatSper and Hv1

The cryoprotective agent (CPA) is one of the most important factors that affects the cryosurvival of sperm. The aim of the present study was to compare two different CPAs, glycerol (Gly) and ethylene glycol (EG), on the cryopreservation of cynomolgus macaques sperm and evaluate the effects of cryopreservation on sperm motility, acrosomal integrity, DNA integrity, mitochondrial function and the sperm membrane ion channels CatSper and Hv1. Compared to fresh sperm, cryopreservation with either 0.7 M Gly or EG decreased the sperm motility (79.8 ± 1.5% Vs. 47.3 ± 1.8% and 47.6 ± 1.4%), acrosomal integrity (89.6 ± 1.2% Vs. 80.1 ± 1.8% and 79.6 ± 1.7%), DNA integrity (91.9 ± 0.7% Vs. 82.9 ± 1.0% and 82.3 ± 1.0%) and mitochondrial membrane potential (87.9 ± 1.8% Vs. 70.6 ± 2.7% and 67.9 ± 2.5%) and the quantity of the CatSper and Hv1 channels determined by Western Blot (p < 0.05), and EG showed equal cryoprotection to cynomolgus sperm in all of the sperm parameters. Our results indicated, for the first time, that cryopreservation decreases the quantity of sperm membrane ion channels (CatSper and Hv1), which might be one of the reasons that frozen sperm have a low fertilizing ability. The study will be beneficial to understand the biological process involved in sperm cryopreservation of nonhuman primates and contribute to improving cryopreservation protocols than can maintain sperm function and fertilizing ability.

Dry Preservation of Spermatozoa: Considerations for Different Species.

The current gold standard for sperm preservation is storage at cryogenic temperatures. Dry preservation is an attractive alternative, eliminating the need for ultralow temperatures, reducing storage maintenance costs, and providing logistical flexibility for shipping. Many seeds and anhydrobiotic organisms are able to survive extended periods in a dry state through the accumulation of intracellular sugars and other osmolytes and are capable of returning to normal physiology postrehydration. Using techniques inspired by nature's adaptations, attempts have been made to dehydrate and dry preserve spermatozoa from a variety of species. Most of the anhydrous preservation research performed to date has focused on mouse spermatozoa, with only a small number of studies in nonrodent mammalian species. There is a significant difference between sperm function in rodent and nonrodent mammalian species with respect to centrosomal inheritance. Studies focused on reproductive technologies have demonstrated that in nonrodent species, the centrosome must be preserved to maintain sperm function as the spermatozoon centrosome contributes the dominant nucleating seed, consisting of the proximal centriole surrounded by pericentriolar components, onto which the oocyte's centrosomal material is assembled. Preservation techniques used for mouse sperm may therefore not necessarily be applicable to nonrodent spermatozoa. The range of technologies used to dehydrate sperm and the effect of processing and storage conditions on fertilization and embryogenesis using dried sperm are reviewed in the context of reproductive physiology and cellular morphology in different species.

Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.

Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane.

Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity.

Cholesterol-Loaded Cyclodextrin Increases the Cholesterol Content of Goat Sperm to Improve Cold and Osmotic Resistance and Maintain Sperm Function after Cryopreservation.

The success of semen cryopreservation depends on sperm membrane integrity and function after thawing. Cholesterol-loaded cyclodextrin (CLC) is used for in vitro incorporation of cholesterol to protect cells against cold temperatures. We hypothesized that CLC treatment also enhances sperm cholesterol content to increase tolerance to osmotic shock and cryoresistance, thereby improving fertility. We confirmed the fact that treatment of goat semen with 3 mg/ml CLC increases sperm cholesterol content using both the Liebermann-Burchard approach and filipin III labeling of membrane cholesterol. Sperm were then treated with or without CLC and cryopreserved. After thawing, sperm cholesterol dramatically fell, even in the presence of CLC, which explains the mechanism of cryocapacitation. CLC treatment, however, maintained a normal prefreeze cholesterol level in sperm after cryopreservation. Furthermore, fresh sperm treated with CLC and subjected to either cold shock or incubated in hypo-, iso-, and hyperosmotic media, designed to mimic stresses associated with freezing/thawing, displayed increased temperature and osmotic tolerance. CLC treatment also improved sperm viability, motility, and acrosome integrity after thawing. Furthermore, CLC treatment did not affect the sperm's ability to undergo in vitro capacitation according to chlortetracycline fluorescence and protein tyrosine phosphorylation. A pilot field trial demonstrated that artificial insemination with sperm that underwent increased cholesterol levels following CLC treatment yielded higher fertility ( ITALIC! P< 0.1) and proliferation ( ITALIC! P< 0.05) rates in vivo than untreated semen from the same ejaculate samples. These observations suggest that CLC treatment could be used to improve cryoprotection during the freezing and thawing of goat sperm.

Effect of tubal explants and their secretions on bovine spermatozoa: modulation of ROS production and DNA damage

Although low levels of reactive oxygen species (ROS) play a physiological role in maintaining sperm function, an increase in ROS generation above these levels may result in the induction of sperm membrane and DNA damage. The main objective of this study was to determine whether bovine oviducal explants (TU) and their conditioned media (CM) have a modulatory effect on the production of ROS, and consequently, on sperm DNA integrity. Thawed sperm were exposed to bovine TU and to CM obtained from the ampullar and isthmal regions after 4 and 12h, and DNA damage and intracellular ROS production was assessed by TUNEL and DHE and SYTOX Green, respectively. Co-incubation of spermatozoa with oviducal explants from the ampullar region (TUa) for 4h resulted in a statistically significant increase in the percentage of spermatozoa with DNA damage compared with controls (P=0.0106), and this increase was positively correlated with ROS levels. Conversely, although the incubation of spermatozoa with explants and conditioned media from the isthmal region (TUi and CMi, respectively) for 12h resulted in an increase of spermatozoa with DNA damage compared with controls (P<0.0001), this increase was not correlated with ROS levels. In conclusion, significant oxidative stress may take place in the oviduct, particularly during short-term incubation, and this may be related to changes in the antioxidant factors present in the oviducal cells and secretions. A redox imbalance in pro-oxidants and antioxidants in the oviduct may lead to oxidative stress and sperm DNA damage.

Factors impacting equine sperm recovery rate and quality following cushioned centrifugation

Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P<0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P<0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400×g or 600×g were applied (90 and 90%, respectively; P>0.05), and no resulting differences in semen quality were detected between these treatment groups (P>0.05). The type of iodixanol cushion medium used (i.e., OptiPrep™, Eqcellsire® Component B, or Cushion Fluid™) did not impact post-centrifugation semen quality, based on the laboratory values measured (P>0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal.