ProAKAP4 as Novel Molecular Marker of Sperm Quality in Ram: An Integrative Study in Fresh, Cooled and Cryopreserved Sperm.
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ABSTRACT:
SUBMITTER: F Riesco M
PROVIDER: S-EPMC7408074 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cryopreservation generates a substantial quantity of ROS in semen, leading to a decline in sperm quality and fertilization capacity. The objective of this study was to investigate the effects of resveratrol and its optimal concentration on ram sperm quality after cryopreservation. Ram semen was diluted with a freezing medium containing different concentrations of resveratrol (0, 25, 50, 75, and 100 μM). After thawing, various sperm parameters such as total motility, progressive motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, glutathione (GSH) content, glutathione synthase (GPx) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, lipid peroxidation (LPO) content, malondialdehyde (MDA) content, ROS level, SIRT1 level, DNA oxidative damage, and AMPK phosphorylation level were assessed. In addition, post-thaw sperm apoptosis was evaluated. Comparatively, the addition of resveratrol up to 75 μM significantly improved the sperm motility and sperm parameters of cryopreserved ram sperm. Specifically, 50 μM resveratrol demonstrated a notable enhancement in acrosome and plasma membrane integrity, antioxidant capacity, mitochondrial membrane potential, adenosine triphosphate (ATP) content, SIRT1 level, and AMPK phosphorylation levels compared to the control group (p < 0.05). It also significantly (p < 0.05) reduced the oxidative damage to sperm DNA. However, detrimental effects of resveratrol were observed at a concentration of 100 μM resveratrol. In conclusion, the addition of 50 μM resveratrol to the cryopreservation solution is optimal for enhancing the quality of cryopreserved ram sperm.
| S-EPMC10740518 | biostudies-literature
Project description:Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa Study design, size, duration (max. 75 words): Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods (max. 75 words): The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance (max. 200 words): The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). The storage time of cryopreserved human spermatozoa does not affect pathways involved in fertility. Study design, size, duration This study included 24 normospermic men after their written informed consent, of whom 13 had cryostored semen for a short-time (1 week) and 11 had cryostored semen for a long-time (median 9 years, range 7-10). RNA was extracted from each frozen-thawed sperm sample and randomized into pools (4 pools for short-time samples and 3 pools for long-time samples), each of 5-6 patients. Wide-transcriptome comparison of short- and long-time pools were analyzed. Participants/materials, setting, methods The study population had a median age of 34.5 years (range: 22-46), without significant difference between the two groups (p-value = 0.147). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The unpaired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance Comparison of gene expression profiles between thawed sperm after a short or long storage period in liquid nitrogen identified 5 unique transcripts significantly upregulated in the latter group (DCP1A, FAM217A, HUWE1, MSI2, PCSKIN). Functional annotation enrichment has shown that the dysregulated transcripts are involved in methylation and protein binding. In particular, HUWE1 encodes an E3 ubiquitin protein ligase required for protein ubiquitination. The protein encoded by DCP1A is a decapping enzyme, necessary for the degradation of mRNAs. The RNA binding protein MSI2 regulates the expression of target mRNAs at the translation level. Interestingly, HUWE1 serves as a central node in cellular stress responses, cell growth and death, signal transduction, etc. During early embryonic development, siRNA-mediated knockdown of HUWE1 induced apoptotic cell death and inhibited normal embryonic development. Consistent with this finding, decreased HUWE1 staining in human embryos was found to be associated with poor embryo quality, suggesting that HUWE1 plays important roles in promoting embryonic development. DCP1A is also essential for embryonic development. Overall, no critical pathways involved in fertility are dysregulated during storage of sperm in liquid nitrogen and upregulation of genes important for early embryonic development may compensate for sperm cryoinjury.
2024-04-09 | GSE225320 | GEO
Project description:Testicular ultrasound is a non-invasive technique that could be very useful for predicting ram seminal quality. Recent software developments allow macroscopic and microscopic evaluation of testicular parenchyma. Thus, the aim of this study was to evaluate the testicular echotexture using ultrasound-video analysis and investigate its possible correlation with semen quality. Nine rams were evaluated for one year using a portable ultrasound scanner and the echotexture was analyzed with ECOTEXT® software. The number of black (Ec1), white (Ec2), and grey pixels (Ec3), tubular density (TD), lumen area (LA), and lumen diameter (LD) were analyzed. Semen was collected by an artificial vagina the same day and the sperm concentration, morphology, motility, viability, phosphatidylserine (PS) translocation, reactive-oxygen-species (ROS) levels, DNA damage and capacitation state were evaluated. Ec2 and Ec3 correlated positively with "bad quality" sperm parameters (the percentage of spermatozoa with high ROS levels, with PS translocation and proximal cytoplasmic droplets), and negatively with motility. In contrast, TD and LA showed a positive correlation with "good quality" parameters (motility or normal morphology) and a negative correlation with spermatozoa with high ROS levels, with DNA fragmentation, and proximal or distal cytoplasmic droplets. Thus, echotexture analysis by ultrasound-video analysis could be a valuable tool for assessing ram fertility.
| S-EPMC8869597 | biostudies-literature
Project description:BackgroundSperm quality at cancer diagnosis is often compromised by the disease and any given gonadotoxic treatment will further diminish fertility.ObjectivesHere, we aim to analyze the cryopreserved sperm quality according to the cancer types as well as the fertility outcomes.MethodsOur study included all cancer patients who cryopreserved sperm over 20 years at Erasme Hospital Brussels (from 1999 to 2019). First sperm samples from 111 hematologic, 104 testicular, 19 prostate, 28 gastrointestinal, and 16 neurological cancer patients were compared.ResultsOligozoo-asthenozoospermia was observed in 30% of the samples, including 19.33% with severe oligozoospermia (<5 million/ml). Our results showed a significant reduction in sperm concentration among testicular cancer (p < 0.01). No significant differences in progressive motility, sperm volume, and number of frozen straws were observed. Significant correlations were found between sperm concentration and cancer type (p <0.01) as well as patients' age (p <0.01). Twenty-eight cancer survivors returned for using their cryopreserved sperm (9.33%), fertilization rate was 60.5% and implantation rate was 29.6%. There was no correlation between sperm concentration and fertility outcomes.ConclusionOur results confirm the negative impact of cancer on sperm quality without affecting assisted reproductive technology (ART) success rate, which is utterly important as a male reproductive health perspective. All cancer patients should be counselled and offered fertility preservation options as a gold standard.
| S-EPMC9297376 | biostudies-literature
Project description:The purpose of this study was to examine the effect of different rutin concentrations on rams epididymal sperm. A local slaughterhouse provided 50 pair of testes from 25 rams. The testes were sent to the lab at room temperature. Spermatozoa were extracted by suspending portions of cauda epididymis in tris solution. Ram sperm was cryopreserved (in liquid nitrogen) in a tris extender containing rutin at 0, 0.5, 0.75, 1, and 1.25 mM. Rutin showed superior sperm total and progressive motility, beat cross frequency, straight line velocity, velocity average pathway and membrane integrity values at 0.75 and 1 mM. The morphology of the sperm and the superoxide dismutase levels did not significantly change with different treatments. Moreover, rutin at 0.75 and 1 mM was also shown to have the highest level of mitochondrial activity. The results showed ATP, total antioxidant capacity, and glutathione peroxidase levels were significantly greater in the rutin 0.75 and 1 mM groups (P < 0.05). Rutin at 0.75 and 1 mM levels had the lowest reactive oxygen species concentrations. Rutin at 0.75 and 1 mM substantially increased the proportion of viable sperm (P < 0.05). The lowest amount of apoptosis was observed in 0.75 and 1 mM rutin. Rutin at 0.75 and 1 mM yielded the least significant percentage of dead sperm. It may be inferred that adding 0.75 and 1 mM to the sperm extender can enhance the quality of the epididymal sperm in rams after the cryopreservation process.
| S-EPMC10322927 | biostudies-literature
Project description:Gene expression profiling of human fresh and cryopreserved sperm
| PRJNA934956 | ENA
Project description:As structural components of sperm, tektins are thought to play a fundamental role in sperm flagellar motility. In this study, Tektin-4 (Hdh-TEKT4) gene was successfully cloned and characterized from the testis tissue in Pacific abalone, Haliotis discus hannai. The full-length cDNA of Hdh-TEKT4 was 1,983 bp, with a coding region of 1,350 bp encoding 51.83 kDa putative protein of 449 deduced amino acids. Hdh-TEKT4 contains a tektin domain including a nonapeptide signature motif (RPGVDLCRD). Fluorescence in situ hybridization revealed that Hdh-TEKT4 localized in the spermatids of Pacific abalone testis. qRT-PCR analysis showed that Hdh-TEKT4 was predominantly expressed in testis tissues. Hdh-TEKT4 mRNA expression was upregulated during the fully mature testicular developmental stage in both seasonal development and EAT exposed abalone. Furthermore, mRNA expression of Hdh-TEKT4 was significantly higher in sperm with higher motility than in sperm with lower motility during peak breeding season, induced spawning activity stages, and after cryopreservation in different cryoprotectants. Taken together, these results indicate that the expression of Hdh-TEKT4 in Pacific abalone sperm might have a positive correlation with sperm motility.
| S-EPMC9081794 | biostudies-literature
Project description:BackgroundRam spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters.ResultsEjaculates were collected using the artificial vagina from four Chal rams and three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen for 15 min and plunged into liquid nitrogen. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics.ConclusionsControlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing.
| S-EPMC3349620 | biostudies-literature
Project description:Testicular cancer (TC) represents the most common cancer affecting men within the reproductive age and is often accompanied by major disturbances in semen parameters. Cryopreservation is recommended in these patients before initiating cancer treatment. Currently, there are no studies reporting the molecular mechanisms associated with altered semen quality in these men. The main objective of this study was to compare the sperm proteome of normozoospermic (motility >40%) and asthenozoospermic (motility <40%) TC patients with normozoospermic infertile men without cancer (control group). Pooled sperm samples from normozoospermic (n = 20), asthenozoospermic (n = 11) TC, and a control group (n = 9) were used for quantitative global proteomic profiling using liquid chromatography-tandem mass spectrometry. A total of 1085, 846, and 982 proteins were identified in normozoospermic TC, asthenozoospermic TC, and control groups, respectively. Functional analysis revealed mitochondrial dysfunction and altered cellular pathways in both normozoospermic and asthenozoospermic TC patients. Comparison of pathway analysis showed no significant difference in fertility-associated proteins/mechanism between the normozoospermic TC patients and infertile men. Western blot analysis revealed under-expression of NDUFS1 associated with mitochondrial dysfunction and overexpression of CD63 involved in sperm maturation in both normozoospermic and asthenozoospermic TC patients. Our proteomic results confirm that defective cellular pathways are associated with reproductive functions in both normozoospermic and asthenozoospermic TC patients before the start of cancer treatment.
| S-EPMC6387327 | biostudies-literature
Project description:Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2 ⋅-), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2 ⋅- and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.
| S-EPMC8138477 | biostudies-literature