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Sperm function, mitochondrial activity and in vivo fertility are associated to their mitochondrial DNA content in pigs

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Journal of Animal Science and Biotechnology 2024年第4期15卷 1550-1558页

作者： marc Llavanera Yentel Mateo‑Otero Estel Vinolas‑Vergés Sergi Bonet marc yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGirona ES‑17003Spain Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGirona ES‑17003Spain Catalan Institution for Research and Advanced Studies(ICREA) Barcelona ES‑08010Spain

Background Despite their low abundance in sperm, mitochondria have diverse functions in this cell type, includ-ing energy production, signalling and calcium regulation. In humans, sperm mitochondrial DNA content(mtDNAc) has been reported to be negatively linked to sperm function and fertility. Yet, the association between mtDNAc and sperm function in livestock remains unexplored. For this reason, this study aimed to shed some light on the link between mtDNAc and sperm function and fertilising potential in pigs. A qPCR method for mtDNAc quantification was optimised for pig sperm, and the association of this parameter with sperm motility, kinematics, mitochondrial activity, and fertility was subsequently *** First, the q PCR method was found to be sensitive and efficient for mtDNAc quantification in pig sperm. By using this technique, mtDNAc was observed to be associated to sperm motility, mitochondrial activity and in vivo, but not in vitro, fertility outcomes. Specifically, sperm with low mtDNAc were seen to exhibit greater motility but decreased mitochondrial activity and intracellular reactive oxygen species. Interestingly, samples with lower mtD-NAc showed higher conception and farrowing rates, but similar in vitro fertilisation rates and embryo development, when compared to those with greater *** These findings enrich our comprehension of the association of mtDNAc with sperm biology, and lay the foundation for future research into employing this parameter as a molecular predictor for sperm function and fer-tility in livestock.

关键词： Fertility Mitochondria Motility mtDNA copy number qPCR Sperm

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

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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 *** 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 ***,whether disparities in the DNA integrity and chromatin condensation and pro-tamination of their sperm exist has not been *** 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;P<0.01);this index,in addition,was observed to be correlated to the sperm concentration of each fraction(Rs=0.973;P<0.001).Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the *** 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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Expression of miR‑138 in cryopreserved bovine sperm is related to their fertility potential

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Journal of Animal Science and Biotechnology 2023年第6期14卷 2383-2394页

作者： Albert Salas‑Huetos Jordi Ribas‑Maynou Yentel Mateo‑Otero Carolina Tamargo marc Llavanera marc yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGirona 17003Spain. Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGirona 17003Spain. Department of Nutrition Harvard T.H.Chan School of Public HealthHarvard UniversityBostonMA 02115USA. Consorcio CIBER M.P.Fisiopatologia de la Obesidad y Nutricion(CIBERobn)Instituto de Salud Carlos III(ISCIII)Madrid 28029Spain. Present Address:Unit of Preventive Medicine and Public Health Faculty of Medicine and Health SciencesUniversitat Rovira i VirgiliReus 43201Spain. Department of Animal Selection and Reproduction The Regional Agri-Food Research and Development Service of Asturias(SERIDA)Gijon 33394Spain. Catalan Institution for Research and Advanced Studies(ICREA) Barcelona 08010Spain.

Background MicroRNAs(miRNAs)are small,single-stranded,non-coding RNA molecules of 22–24 nucleotides that regulate gene *** the last decade,miRNAs have been described in sperm of several mammals,including *** is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans;however,little is known about the content,expression,and function of sperm-borne miRNAs in early bovine *** this study,total sperm RNA was isolated from 29 cryopreserved sperm samples(each coming from a separate bull)using a RNeasy kit and treatment with DNase *** concentration and purity were determined through an Epoch spectrophotometer and an Agilent *** expression of 10 candidate miRNAs in bovine sperm(bta-miR-10a,bta-miR-10b,bta-miR-138,bta-miR-146b,bta-miR-19b,bta-miR-26a,bta-miR-34a,bta-miR-449a,bta-miR-495 and btamiR-7),previously identified in testis and/or epididymis,was evaluated with *** cel-miR-39-3p was used as a spike-in exogenous *** Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility[HF;non-return rates(NRR)ranging from 39.5 to 43.5]and those with subfertility(SF;NRR ranging from 33.3 to 39.3).Several sperm functionality parameters(e.g.,viability,membrane stability or oxygen consumption,among others)were measured by multiplexing flow cytometry and oxygen sensing *** RNA concentration and purity(260/280 nm ratio)(mean±SD)from the 29 samples were 99.3±84.6 ng/μL and 1.97±0.72,*** results confirmed the lack of RNA from somatic *** terms of the presence or absence of miRNAs,and after applying the Livak method,8 out of 10 miRNAs(bta-miR-10b,-138,-146b,-19b,-26a,-449a,-495,-7)were consistently detected in bovine sperm,whereas the other two(bta-miR-10a,and-34a)were ***,the relative expression of one miRNA(bta-miR-138)in sperm was significantly lower in the SF than in the HF gr

关键词： Bovine Fertility miRNAs Sperm

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Sperm chromatin condensation as an in vivo fertility biomarker in bulls:a flow cytometry approach

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Journal of Animal Science and Biotechnology 2022年第2期13卷 418-429页

作者： marc Llavanera Jordi Ribas-Maynou Ariadna Delgado-Bermúdez Sandra Recuero Rodrigo Muiño Carlos OHidalgo Carolina Tamargo Sergi Bonet Yentel Mateo-Otero marc yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Faculty of SciencesUniversity of GironaC/Maria Aurèlia Campany69ES-17003 GironaSpain Department of Biology Unit of Cell BiologyFaculty of SciencesUniversity of GironaES-17003 GironaSpain Department of Animal Pathology Faculty of Veterinary MedicineUniversity of Santiago de CompostelaES-15705 LugoSpain Department of Animal Selection and Reproduction The Regional Agri-Food Research and Development Service of Asturias(SERIDA)E-33394 GijónSpain

Background:Genetic selection in cattle has been directed to increase milk ***,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the ***,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive *** humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male *** objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in *** study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein *** outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow *** quality parameters(morphology,viability,total and progressive motility)were also *** correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo *** morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile ***,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in ***,the percentage of viable sperm with poor chromatin condensation showed better accura

关键词： Bull Chromatin Chromomycin A3 Condensation Fertility Flow cytometry Sperm

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Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers

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Journal of Animal Science and Biotechnology 2022年第2期13卷 473-487页

作者： Yentel Mateo-Otero Pol Fernández-López Ariadna Delgado-Bermúdez Pau Nolis Jordi Roca Jordi Miró Isabel Barranco marc yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaES-17003 GironaSpain Department of Biology Unit of Cell BiologyFaculty of SciencesUniversity of GironaES-17003 GironaSpain Centre d’Estudis Avançats de Blanes(CEAB) Spanish Research Council(CSIC)ES-17300 GironaSpain Magnetic Nuclear Resonance Facility Autonomous University of BarcelonaBellaterraES-08193 Cerdanyola del VallèsSpain Department of Animal Medicine and Surgery Faculty of Veterinary MedicineUniversity of MurciaES-30100 MurciaSpain Equine Reproduction Service Department of Animal Medicine and SurgeryFaculty of Veterinary MedicineAutonomous University of BarcelonaBellaterraES-08193 Cerdanyola del VallèsSpain Department of Veterinary Medical Sciences University of BolognaIT-40064 Ozzano dell’EmiliaBolognaItaly

Background:Metabolomic approaches,which include the study of low molecular weight molecules,are an emerging-omics technology useful for identification of *** this field,nuclear magnetic resonance(NMR)spectroscopy has already been used to uncover(in)fertility biomarkers in the seminal plasma(SP)of several mammalian ***,NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried ***,this study aimed to evaluate the putative relationship between SPmetabolites and in vivo fertility *** this end,24 entire ejaculates(three ejaculates per boar)were collected from artificial insemination(AI)-boars throughout a year(one ejaculate every 4 months).Immediately after collection,ejaculates were centrifuged to obtain SP-samples,which were stored for subsequent metabolomic analysis by NMR *** outcomes from 1525 inseminations were recorded over a year,including farrowing rate,litter size,stillbirths per litter and the duration of ***:A total of 24 metabolites were identified and quantified in all *** operating characteristic(ROC)curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate(area under the curve[AUC]=0.764)while carnitine(AUC=0.847),hypotaurine(AUC=0.819),sn-glycero-3-phosphocholine(AUC=0.833),glutamate(AUC=0.799)and glucose(AUC=0.750)showed it for litter ***,citrate(AUC=0.743),creatine(AUC=0.812),phenylalanine(AUC=0.750),tyrosine(AUC=0.753)and malonate(AUC=0.868)levels had discriminative capacity for stillbirths per litter;and malonate(AUC=0.767)and fumarate(AUC=0.868)levels for gestation ***:The assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in ***,supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their f

关键词： Artificial insemination in vivo fertility Metabolomics NMR Pregnancy outcomes Seminal plasma

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Determination of double-and single-stranded DNA breaks in bovine sperm is predictive of their fertilizing capacity

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Journal of Animal Science and Biotechnology 2023年第1期14卷 134-151页

作者： Jordi Ribas‑Maynou Ariadna Delgado‑Bermúdez Yentel Mateo‑Otero Estel Vinolas Carlos O.Hidalgo WSteven Ward marc yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Faculty of SciencesInstitute of Food and Agricultural TechnologyUniversity of GironaC/Maria Aurèlia Campany69ES‑17003 GironaSpain. Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaES‑17003 GironaSpain. Institute for Biogenesis Research Department of AnatomyBiochemistry&PhysiologyJohn A.Burns School of MedicineUniversity of Hawaii at ManoaHonoluluHI 96822USA. Department of Animal Selection and Reproduction The Regional Agri-Food Research and Development Service of Asturias(SERIDA)ES‑33394 GijónSpain. Catalan Institution for Research and Advanced Studies(ICREA) ES‑08010 BarcelonaSpain.

Background:The analysis of chromatin integrity has become an important determinant of sperm *** frozenthawed bovine sperm,neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well *** aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics,and to assess if this parameter can predict pregnancy rates in ***:A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods(0-2 h and 2-4 h),analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays,chromatin deprotamination and decondensation,sperm motility,viability,acrosomal status,and intracellular levels of total ROS,superoxides and *** of 59,605 females was conducted using sperm from the same bulls,thus obtaining the non-return to estrus rates after 90 d(NRR).Results showed an increased rate of double-stranded breaks in the first period(0-2 h:1.29±1.01%/h vs.2-4 h:0.13±1.37%/h;P0.05).Finally,whereas no correlations between bull fertility and any dynamic parameter were found,there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet(Rs=-0.563,P=0.003),between NRR and basal progressive motility(Rs=0.511,P=0.009),and between NRR and sperm with high ROS at 4 h post-thaw(Rs=0.564,P=0.003).Conclusion:The statistically significant correlations found between intracellular ROS,sperm viability,sperm motility,DNA damage

关键词： Cattle Chromatin Comet test DNA damage Fertility Sperm Sperm quality

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GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

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Journal of Animal Science and Biotechnology 2019年第4期10卷 931-941页

作者： marc Llavanera Ariadna Delgado-Bermúdez Beatriz Fernandez-Fuertes Sandra Recuero Yentel Mateo Sergi Bonet Isabel Barranco marc yeste Biotechnology of Animal and Human Reproduction (Techno Sperm) Unit of Cell Biology Department of Biology Institute of Food and Agricultural Technology University of Girona

Background: Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination(AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments,which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm(i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo. In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine ***: Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar ***, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor(PFE) than in good(GFE) freezability ejaculates in both pre-frozen(1.00 INT·mm^2± 0.14 INT·mm^2 vs.0.72 INT·mm^2± 0.15 INT·mm^2;P < 0.05) and post-thawed(0.96 INT·mm^2± 0.20 INT·mm^2 vs. 70 INT·mm^2± 0.19 INT·mm^2;P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species(ROS) production, and high membrane lipid disorder postthaw(P < 0.05).Conclusions: The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar

关键词： Boar Cryopreservation GSTM3 IZUMO1 ROS Sperm

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New insights into SET protein during mouse spermatogenesis

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Asian Journal of Andrology 2014年第5期16卷 783-783页

作者： marc yeste The State Key Laboratory of Reproductive Medicine Clinical Center of Reproductive Medicine First Affiliated Hospital Nanjing Medical University Nanjing China

Spermatogenesis is a complex, unique process that takes place in the testis. It consists of a set of events including mitotic （from spermatogonia to primary spermatocytes） and meiotic divisions （from primary spermatocytes to early round spermatids）, and spermiogenesis, a differentiation step from early round spermatids to testicular spermatozoa Although spermatogenesis is known as a well autocrine- and paracrine-regulated process, some proteins involved in its regulation remain to be determined. Related to this, the recent study conducted by Dai et al. have shed light on the relevant function of SET protein, a protein phosphatase 2A （PP2A） inhibitor, also known as I2PP2A, or TAF-Ib. This protein, identified for the first time in 1992, has been described to be involved in multiple cell functions, such as control of cell cycle and apoptosis, gene transcription, epigenetic regulation, and nucleosome assembly. Apart from these functions, it is worth noting that SET has been to be involved in the regulation found in theca cells, where appears of androgen biosynthesis, and in mature oocytes, where plays a key role in the segregation of sister chromatids during the second meiotic division SET protein inhibits PP2A by enhancing lyase activity of P450c17, a substrate for PP2A when it is Serand Thr-phosphorylated. PP2A function in reproductive physiology is not restricted to gamete production （i.e. spermatogenesis and oogenesis）, but this protein is also involved in human sperm capacitation.

关键词： New insights SET protein Spermatogenesis

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Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

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Journal of Animal Science and Biotechnology 2020年第1期11卷 20-31页

作者： Ariadna Delgado-Bermúdez marc Llavanera Leira Fernández-Bastit Sandra Recuero Yentel Mateo-Otero Sergi Bonet Isabel Barranco Beatriz Fernández-Fuertes marc yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Unit of Cell BiologyDepartment of BiologyFaculty of SciencesInstitute of Food and Agricultural TechnologyUniversity of GironaC/Maria Aurèlia Campany69Campus MontiliviE-17003 GironaSpain

Background:Aquaporins(AQPs)are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs)and super ***3,AQP7,AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and *** exchange is an important event in cryopreservation,which is the most efficient method for long-term storage of ***,the freezethaw process leads to sperm damage and a loss of fertilizing *** that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process,AQPs might play a crucial role in boar semen *** this context,the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different ***:Inhibition of different groups of AQPs was found to have different effects on boar sperm *** the use of 1,3-propanediol(PDO),an inhibitor of orthodox AQPs and GLPs,decreased total motility(P<0.05),it increased post-thaw sperm viability,lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P<0.05).When acetazolamide(AC)was used as an inhibitor of orthodox AQPs,the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P<0.05).Finally,the addition of phloretin(PHL),a GLP inhibitor,had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P<0.05).Conclusions:The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm ***,the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

关键词： Acetazolamide Aquaporins Boar Phloretin Propanediol Sperm

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ABTL0812 enhances antitumor effect of paclitaxel and reverts chemoresistance in triple-negative breast cancer models

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Cancer Communications 2022年第6期42卷 567-571页

作者： Emma Polonio-Alcala Sònia Solé-Sánchez Pau Munoz-Guardiola Elisabet Megías-Roda Héctor Perez-Montoyo marc yeste-Velasco Jose Alfón Jose Miguel Lizcano Carles Domènech Santiago Ruiz-Martínez Teresa Puig New Therapeutic Targets Laboratory(TargetsLab)-Oncology Unit Department of Medical SciencesUniversity of GironaGirona 17003Spain Product Process and Production Engineering Research Group(GREP)Department of Mechanical Engineering and Industrial ConstructionUniversity of GironaGirona 17003Spain Ability Pharmaceuticals SLCerdanyola del VallesBarcelona 08290Spain Protein Kinases and Signal Transduction Laboratory Department de Bioquimica i Biologia Molecular and Institut de NeurocienciesUniversitat Autonoma de BarcelonaBellaterraBarcelona 08193Spain

Dear Editor,Triple-negative breast cancer(TNBC)accounts for 20%of all breast carcinomas and lacks a validated targeted therapy;thus,currently,cytotoxic chemotherapy is the treatment of choice[1].Compared with other breast cancer types,patientswith TNBCare younger,have larger tumors,a higher risk of metastasis,and a higher rate of recurrence[2].Among all the subtypes described by Lehmann et al.[3],mesenchymal-like and mesenchymal stem-like(MSL)subtypes had the lowest 5-year distant metastasis-free survival rates.

关键词： ABTL0812 TRIB3 Autophagy Breast Cancer Targeted Therapy Chemoresistance

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