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International Journal of Molecular... Jun 2024Proteasome 26S Subunit, Non-ATPase 9 () plays an important role in the balance of protamine and the stability of the nucleolar structure during spermatogenesis. In this...
Proteasome 26S Subunit, Non-ATPase 9 () plays an important role in the balance of protamine and the stability of the nucleolar structure during spermatogenesis. In this study, we cloned the of and analyzed its expression pattern. was identified on the Z chromosome of , which is considered an interesting candidate gene for spermatogenesis. qRT-PCR and FISH experiments showed that the gene was significantly highly expressed in the testes. It is worth noting that the expression level of in male fish testes is significantly higher than that in pseudomales. In order to further explore the role of in spermatogenesis, a male testicular cell line was used as the experimental material. The results of the -RNAi and overexpression experiments showed that had a synergistic effect with spermatogenesis-related genes , , and , but had an antagonistic effect with . Our findings offer a scientific foundation for comprehending the role of in the spermatogenesis regulatory network of .
Topics: Animals; Spermatogenesis; Male; Testis; Sex Chromosomes; Fish Proteins; Proteasome Endopeptidase Complex; Cloning, Molecular
PubMed: 38928079
DOI: 10.3390/ijms25126372 -
PloS One 2024Melatonin (MEL) is an indole amine molecule primarily produced in the pineal gland. Melatonin has been shown in numerous studies to have antifibrotic effects on the...
BACKGROUND
Melatonin (MEL) is an indole amine molecule primarily produced in the pineal gland. Melatonin has been shown in numerous studies to have antifibrotic effects on the kidney, liver, and other organs. However, it is still unclear how melatonin works in bladder fibrosis. We explored how melatonin affects animals with bladder fibrosis and the underlying mechanisms.
MATERIALS AND METHODS
MEL was used to treat human bladder smooth muscle cells (HBdSMCs) after they were stimulated with transforming growth factor-β1 (TGF-β1) in vitro. Proteomic analysis and bioinformatic analysis of the altered expression of these proteins were subsequently performed on HBdSMCs from the different processing methods. To construct an in vivo bladder fibrosis model, we injected protamine sulfate (PS) and lipopolysaccharide (LPS) twice a week into the rat bladder for six weeks. After two weeks of PS/LPS treatment, the mice in the treatment group were treated with MEL (20 mg/kg/d) for 4 weeks. Finally, we detected the expression of fibrosis markers from different perspectives. The TGF-β1/Smad pathway and epithelial-mesenchymal transition (EMT) in cell and bladder tissues were also identified. Further proteomic analysis was also performed.
RESULTS
In vitro, we found that TGF-β1 treatment enhanced the expression of the fibrosis markers collagen III and α-SMA in HBdSMCs. E-cadherin expression decreased while the TGF-β1/Smad pathway was activated. Vimentin and N-cadherin expression was also elevated at the same time. Similar findings were observed in the LPS group. After MEL treatment, the expression of collagen III and α-SMA decreased, the expression of E-cadherin increased, and the expression of vimentin and N-cadherin also decreased. According to our quantitative proteomics analysis, CCN1 and SQLE may be important proteins involved in the development of bladder fibrosis. MEL decreased the expression of these genes, leading to the relief of bladder fibrosis. Bioinformatics analysis revealed that the extracellular space structure related to metabolic pathways, actin filament binding, and stress fibers can serve as a pivotal focus in the management of fibrosis.
CONCLUSION
Melatonin attenuates bladder fibrosis by blocking the TGF-β1/Smad pathway and EMT. CCN1 appears to be a possible therapeutic target for bladder fibrosis.
Topics: Rats; Humans; Mice; Animals; Transforming Growth Factor beta1; Vimentin; Melatonin; Signal Transduction; Urinary Bladder; Lipopolysaccharides; Proteomics; Fibrosis; Epithelial-Mesenchymal Transition; Collagen; Cadherins
PubMed: 38478501
DOI: 10.1371/journal.pone.0295104 -
Poultry Science Aug 2023Arginine is the main amino acid that constitutes the sperm protamine of roosters, named galline, which complexes with sperm DNA, allowing high compaction of its...
Arginine is the main amino acid that constitutes the sperm protamine of roosters, named galline, which complexes with sperm DNA, allowing high compaction of its chromatin. Arginine supplementation has positive effects on semen quality in aged roosters, but this supplementation is not known to limit the progressive worsening of sperm chromatin compaction. This work aimed to verify whether L-arginine supplementation in the feed improve or maintain sperm chromatin quality since aging in roosters is usually accompanied by worsening chromatin quality. Four groups of 52-wk-old Ross AP95 lineage roosters were used, of which 6 semen samples per group were evaluated, totaling 24 samples. Another 24 samples, 6 per group, were evaluated after 6 wk of supplementation when one group was not supplemented (control) and the other 3 were supplemented with 1.15 kg (treatment 1), 2.17 kg (treatment 2), and 3.18 kg (treatment 3) of L-arginine/ton of feed. Computer image analysis of semen smears stained with toluidine blue pH 4.0 was used for sperm chromatin evaluation. Sperm chromatin was evaluated for compaction heterogeneity and compaction intensity by percentage decompaction relative to standard heads and by integrated optical density (IOD), which was used for the first time to identify sperm chromatin changes. Sperm head morphology was also evaluated by means of area and length. The IOD proved to be more efficient in identifying changes in rooster sperm chromatin compaction than the percentual decompaction. In general, chromatin compaction was positively influenced by the supplementation with L-arginine, being better in the supplementation with the highest levels tested. This was corroborated by the smaller average of the variables referring to the size of the spermatozoa heads of the animals that received feed with a higher content of L-arginine, since better compacted heads naturally tend to be smaller. Finally, arginine supplementation was able to limit or even improve sperm chromatin decompaction during the experimental period.
Topics: Male; Animals; Semen; Semen Analysis; Chickens; Diet; Chromatin; Spermatozoa; Dietary Supplements; Arginine; Aging; Sperm Motility
PubMed: 37302332
DOI: 10.1016/j.psj.2023.102805 -
Clinical and Applied... 2024Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is...
INTRODUCTION
Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is commonly used for therapeutic, interventional, and surgical indications. Protamine sulfate (PrSO) is frequently used to neutralize UFH. This study aimed to investigate the comparative neutralization profiles of AA and PrSO for heparins of bovine, ovine, and porcine origin.
MATERIALS AND METHODS
The neutralization effect of PrSO at 25 µg/ml and AA at 100 µg/ml was studied on an approximate surgical/interventional concentration of heparin by supplementing whole blood with each of the heparins at 25 µg/ml. For the clotting profile (activated partial thromboplastin time: aPTT), amidolytic (anti-Xa and anti-IIa), and thrombin generation assay each of the heparin were supplemented from -10-0.62 µg/ml.
RESULTS
In the whole blood ACT studies, all three heparins produced strong anti-coagulant effects (400-450 seconds) compared to saline (130-150 seconds). Both AA and PrSO almost fully neutralized the anti-coagulant effects of heparins (140-160 seconds). Both antidotes completely reversed the anticoagulant effects of all three heparins in the aPTT and thrombin generation assay. However, PrSO was more effective in neutralizing the anti-Xa, and anti-IIa effects than AA, which only partially neutralized these effects.
CONCLUSION
Andexanet alfa at 100 µg/ml effectively neutralizes the therapeutic and surgical/interventional concentrations of heparins in settings. While differences in the anti-Xa, and anti-IIa effects between heparins were noted, anti-coagulant effect of these agents in the aPTT assay were comparable. A similar neutralization profile was observed in the ACT and thrombin generation assays by both agents.
Topics: Recombinant Proteins; Factor Xa; Cattle; Sheep; Swine; Animals; Anticoagulants; Heparin; Protamines; Heparin Antagonists; Blood Coagulation; Thrombin Time
PubMed: 38656136
DOI: 10.1177/10760296241247558 -
Journal of Diabetes May 2024The study aimed to compare glycemic control and pregnancy outcomes in women with type 1 diabetes mellitus (T1DM) using multiple daily injection therapy (MDI) and... (Comparative Study)
Comparative Study
INTRODUCTION
The study aimed to compare glycemic control and pregnancy outcomes in women with type 1 diabetes mellitus (T1DM) using multiple daily injection therapy (MDI) and continuous subcutaneous insulin infusion (CSII) and to compare outcomes of women treated with long-acting insulin or neutral protamine Hagedorn (NPH).
METHODS
This multicenter prospective cohort study involved women with pregestational T1DM treated with MDI and CSII. Primary outcome was glycated hemoglobin (HbA1c) before and during pregnancy. Secondary outcomes included maternal and neonatal outcomes and quality of life.
RESULTS
Of the 121 studied women, the average age was 28.48 years, and the average body mass index was 21.29 kg/m at conception and 26.32 kg/m at delivery. Of the studied women, 78.51% had planned pregnancy. Women treated with MDI and CSII had comparable HbA1c before pregnancy or in the first and second trimesters. In the third trimester, women on CSII therapy had significantly lower HbA1c (6.07 ± 0.62 vs 6.20 ± 0.88%, p = .017), higher HbA1c on-target rate (71.43% vs 64.62%, p = .030), and greater decline of HbA1c from preconception to the third trimester (-0.65 vs -0.30%, p = .047). Fewer daily insulin requirements were observed in those used CSII compared with MDI-treated women (0.60 ± 0.22 vs 0.73 ± 0.25 U/kg/day, p = .004). Newborns born of mothers treated with the CSII method were more likely to have neonatal jaundice (adjusted odds ratio [OR] 2.76, 95% confidence interval [CI] 1.16-6.57) and neonatal intensive care unit (adjusted OR 3.73, 95%CI 1.24-11.16), and women on CSII had lower scores in patient-reported quality of life (p = .045). In the MDI group, those receiving long-acting insulin had nonsignificant lower HbA1c and higher HbA1c on-target rate in the second and third trimesters, compared with those treated with NPH.
CONCLUSIONS
Insulin pump users may achieve better glycemic control than multiple daily insulin injections, which did not substantially improve pregnancy outcome.
Topics: Humans; Female; Pregnancy; Diabetes Mellitus, Type 1; Adult; Insulin; Insulin Infusion Systems; Prospective Studies; Hypoglycemic Agents; Pregnancy in Diabetics; Injections, Subcutaneous; Glycated Hemoglobin; Pregnancy Outcome; Infusions, Subcutaneous; Blood Glucose; Quality of Life; Glycemic Control
PubMed: 38664886
DOI: 10.1111/1753-0407.13558 -
Theriogenology Feb 2024Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present...
Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1/PRM2 with r = +0.6, while PRM1/ACR (r = -0.495), PRM2/ZPBP (r = -0.645) and CRISP3/ACR (r = -0.551) were inversely correlated. In PF direct correlations were registered for PRM1/PRM2 (r = +0.629), PRM1/PRM3 (r = +0.657), PRM2/SPA17 (r = +0.685), SPA17/PLCZ1 (r = +0.786) and PRM3/ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1/PRM2 (r = +0.625), PRM1/PRM3 (r = +0.368); PRM2/SPA17 (r = +0.465), SPA17/PLCZ1 (r = +0.637) and PLCZ1/ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1, that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions.
Topics: Pregnancy; Female; Male; Horses; Animals; Semen; Spermatozoa; Infertility, Male; Semen Preservation; Cryopreservation; Type C Phospholipases; Sperm Motility; Horse Diseases
PubMed: 38142472
DOI: 10.1016/j.theriogenology.2023.12.012 -
Medicine Nov 2023In the process of spermatogenesis and maturation, histones of the sperm nucleus were gradually replaced by protamine. Abnormal sperm nucleoprotein histotype conversion...
In the process of spermatogenesis and maturation, histones of the sperm nucleus were gradually replaced by protamine. Abnormal sperm nucleoprotein histotype conversion can make sperm DNA unstable and affect sperm function. The aim of this study is to investigate the impact of high and low proportion of sperm histone positivity in semen sample on embryonic development and assisted reproductive technology results, and to evaluate its diagnostic value in assisted reproduction. Sperm nuclear status was detected with aniline blue staining. Under acidic conditions, aniline blue combines with histones rich in lysine residues to form blue compounds. The groups were divided according to the critical value of sperm histone positive ratio of 30%. Using the intracytoplasmic sperm injection procedure, the fertilization rate and normal fertilization rate in the normal sperm histone positive ratio group were significantly higher than those in the abnormal group, and the difference was statistically significant (P < .001). Using the in vitro fertilization procedure, the effect of sperm histone positive ratio on each index was not statistically different. Overall the study provides some preliminary evidence that abnormal sperm histones may be a factor that affects the fertilization success of intracytoplasmic sperm injection procedures. However, more research is needed to confirm this finding to determine the exact mechanism by which abnormal sperm histones affect fertilization.
Topics: Pregnancy; Female; Humans; Male; Histones; Chromatin; Semen; Spermatozoa; Fertilization in Vitro; Embryonic Development
PubMed: 38013277
DOI: 10.1097/MD.0000000000036113 -
Diagnostics (Basel, Switzerland) Aug 2023Intraoperative fluid therapy is regularly used in patients undergoing cardiac surgery procedures with cardiopulmonary bypass (CPB). Although fluid administration has...
Intraoperative fluid therapy is regularly used in patients undergoing cardiac surgery procedures with cardiopulmonary bypass (CPB). Although fluid administration has several advantages, it unavoidably leads to hemodilution. The hemodilution may further influence the interpretation of concentration-based laboratory parameters like hemoglobin (Hgb), platelet count (PLT) or prothrombin time (PT). These all parameters are commonly used to guide blood product substitution. To assess the impact of dilution on these values, we performed a prospective observational study in 174 patients undergoing elective cardiac surgery. We calculated the total blood volume according to Nadler's formula, and fluid therapy was correlated with a newly developed dilution coefficient formula at the end of CPB. Intravenously applied fluids were measured from the beginning of the anesthesia (baseline, T) and 15 min after the end of protamine infusion (end of CPB, T). The amount of the administered volume (crystalloids or colloids) was calculated according to the percentage of the intravascular fluid effect, and intraoperative diuresis was further subtracted. The median blood volume increased by 148% in all patients at T compared to the calculated total blood volume at T. This led to a dilution-dependent decrease of 38% in all three parameters (Hgb 24%, corrCoeff = 0.53; PLT 41%, corrCoeff = 0.68; PT 44%, corrCoeff = 0.54). The dilution-correlated decrease was significant for all parameters ( < 0.001), and the effect was independent from the duration of CPB. We conclude that the presented calculation-based approach could provide important information regarding actual laboratory parameters and may help in the guidance of the blood product substitution and potential transfusion thresholds. Further research on the impact of dilution and related decision-making for blood product substitution, including its impact on morbidity and mortality, is warranted.
PubMed: 37568959
DOI: 10.3390/diagnostics13152596 -
Toxics Apr 2024Obesity, a chronic metabolic disorder, is related to cardiovascular diseases, diabetes, cancer, and reproductive disorders. The relationship between obesity and male...
BACKGROUND
Obesity, a chronic metabolic disorder, is related to cardiovascular diseases, diabetes, cancer, and reproductive disorders. The relationship between obesity and male infertility is now well recognized, but the mechanisms involved are unclear. We aimed to observe the effect of obesity on spermatogenesis and to investigate the role of histone ubiquitination and acetylation modifications in obesity-induced spermatogenesis disorders.
METHODS
Thirty male C57BL/6J mice were randomly divided into two groups. The control group was fed with a general maintenance diet (12% fat), while a high-fat diet (HFD) group was fed with 40% fat for 10 weeks; then, they were mated with normal females. The fertility of male mice was calculated, testicular and sperm morphology were observed, and the expression levels of key genes and the levels of histone acetylation and ubiquitination modification during spermatogenesis were detected.
RESULTS
The number of sperm was decreased, as well as the sperm motility, while the number of sperm with malformations was increased. In the testes, the mRNA and protein expression levels of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), chromosome region maintenance-1 protein (CRM1), high-mobility group B2 (HMGB2), phosphoglycerate kinase 2 (PGK2), and testicular angiotensin-converting enzyme (tACE) were decreased. Furthermore, obesity led to a decrease in ubiquitinated H2A (ubH2A) and reduced levels of histone H3 acetylation K18 (H3AcK18) and histone H4 acetylation K5, K8, K12, and K16 (H4tetraAck), which disrupted protamine 1 (Prm1) deposition in testis tissue.
CONCLUSION
These results suggest that low levels of histone ubiquitination and acetylation are linked with obesity-induced disorders during spermatogenesis, contributing to a better understanding of obesity-induced damage to male reproduction.
PubMed: 38668519
DOI: 10.3390/toxics12040296 -
International Journal of Molecular... Feb 2024Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to...
Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI-median fluorescence intensity-and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified.
Topics: Swine; Male; Animals; Chromatin; Flow Cytometry; 8-Hydroxy-2'-Deoxyguanosine; Biofilms; Semen; Bioreactors; Spermatozoa; DNA; DNA Fragmentation; Disulfides; Bridged Bicyclo Compounds
PubMed: 38396632
DOI: 10.3390/ijms25041953