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Chromatin condensation but not dna integrity of pig sperm is greater in the sperm-rich fraction

Journal of Animal Science and Biotechnology 2024年第1期15卷 171-181页

作者： Estel Viñolas-Vergés Jordi Ribas-Maynou Isabel Barranco Camila Peres Rubio Sergi Bonet Jordi Roca Marc Yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGironaSpain Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGironaSpain Department of Medicine and Animal Surgery Faculty of Veterinary ScienceUniversity of MurciaMurciaSpain Catalan Institution for Research and Advanced Studies(ICREA) S08010 BarcelonaSpain

Background Protamination and condensation of sperm chromatin as well as dna integrity play an essential role during fertilization and embryo development.In some mammals,like pigs,ejaculates are emitted in three separate fractions:pre-sperm,sperm-rich(SRF)and post sperm-rich(PSRF).These fractions are known to vary in volume,sperm concentration and quality,as well as in the origin and composition of seminal plasma(SP),with differences being also observed within the SRF one.Yet,whether disparities in the dna integrity and chromatin condensation and pro-tamination of their sperm exist has not been interrogated.Results This study determined chromatin protamination(Chromomycin A3 test,CMA_(3)),condensation(Dibromobi-mane test,DBB),and dna integrity(Comet assay)in the pig sperm contained in the first 10 m L of the SRF(SRF-P1),the remaining portion of the sperm-rich fraction(SRF-P2),and the post sperm-rich fraction(PSRF).While chromatin protamination was found to be similar between the different ejaculate fractions(P>0.05),chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF(P=0.018 and P=0.004,respectively).Regarding dna integrity,no differences between fractions were observed(P>0.05).As the SRF-P1 has the highest sperm concentra-tion and ejaculate fractions are known to differ in antioxidant composition,the oxidative stress index(OSi)in SP,calcu-lated as total oxidant activity divided by total antioxidant capacity,was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF(0.42±0.06 vs.0.23±0.09 and 0.08±0.00,respectively;Pdna integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF.This could be related to the OSi of each fraction.

关键词： Chromatin Condensation dna integrity Ejaculate fractions Pig Protamination Sperm

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Effects of Rosmarinic Acid on dna integrity and H19 Differentially Methylated Region Methylation Levels in Human Sperm Preserved by Freeze-Drying

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Reproductive and Developmental Medicine 2021年第1期5卷 9-14页

作者： Yi-Yu Wang Wei-Jie Zhu Department of Developmental and Regenerative Biology College of Life Science and TechnologyJinan UniversityGuangzhou 510632China

Original Article Effects of Rosmarinic Acid on dna integrity and H19 Differentially Methylated Region Methylation Levels in Human Sperm Preserved by Freeze-Drying Wang Yi-Yu,Zhu Wei-Jie Published 2021-03-25 Cite as Reprod Dev Med,2021,05 Reprod Dev Med,2021,05(1):9-14.DOI:10.4103/2096-2924.309790 Abstract Objective:To investigate the effects of rosmarinic acid(RA)on the dna integrity and methylation levels of the H19 differentially methylated region(DMR)of freeze-dried human sperm after 1 week and 6 months of storage at 4℃.Methods:Semen samples from 15 healthy normospermic donors were used in this study.The samples were divided into five groups,including the control group with fresh sperm and four experimental groups with freeze-dried sperm(1-week storage with EGTA buffer solution,Group A;1-week storage with EGTA buffer solution containing 105μmol/L RA,Group B;6-month storage with EGTA buffer solution,Group C;and 6-month storage with EGTA buffer solution containing 105μmol/L RA,Group D).dna integrity was evaluated using the sperm chromatin dispersion test.H19 DMR methylation levels were detected by bisulfite sequencing polymerase chain reaction.Results:After 1 week of storage,no differences in sperm dna integrity were observed among Groups A,B,and controls(P>0.05).After 6 months of storage,the sperm dna integrity of Group D did not change significantly compared with that of the control group(P>0.05),whereas that of Group C decreased significantly(P0.05).Conclusions:The dna integrity of freeze-dried human sperm can be effectively protected by adding RA within 6 months,and the H19 DMR methylation level of human sperm can be maintained for 6 months after freeze-drying.

关键词： dna integrity dna Methylation Freeze-Drying Human Sperm Rosmarinic Acid

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Sperm cryodamage occurs after rapid freezing phase:flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation

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Journal of Animal Science and Biotechnology 2016年第4期7卷 495-503页

作者： L.S.Castro T.R.S.Hamilton C.M.Mendes M.Nichi V.H.Barnabe J.A.Visintin M.E.O.A.Assumpcao laboratory of spermatozoa biology department of animal reproductionschool of veterinary medicine and animal scienceuniversity of sao pauloBrazil laboratory of in vitro fertilization cloning and animal transgenesisdepartment of animal reproductionschool of veterinary medicine and animal scienceuniversity of sao paulosao pauloBrazil laboratory of andrology.department of animal reproduction school of veterinary medicine and animal scienceuniversity of sao paulosao pauloBrazil

Background： In order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls （n = 4） were separated in 3 treatments： fresh （37 ℃）; cooled （5 ℃）; and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential （MMP） and dna damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry. Results： There was an increase in the percentage of sperm with dna damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters. Conclusion： Based on our results, we conclude that cryopreservation affects cellular and dna integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery （SOD and GPX enzymes） is not enough to control cryodamage.

关键词： Bovine dna integrity JC-1 Sperm viability

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