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Chemoselective labeling-based spermatozoa glycan imaging reveals abnormal glycosylation in oligoasthenotspermia

Chinese Chemical Letters 2024年第4期35卷 475-480页

作者： Lijia Xu Tong Zhong Wei Zhao Bing Yao Lin Ding Huangxian Ju State Key Laboratory of Analytical Chemistry for Life Science School of Chemistry and Chemical EngineeringNanjing UniversityNanjing 210023China Department of Reproductive Medical Center Jinling HospitalSchool of MedicineNanjing UniversityNanjing 210002China Chemistry and Biomedicine Innovation Center(ChemBIC) Nanjing UniversityNanjing 210023China Center of Clinical Laboratory Science The Affiliated Cancer Hospital of Nanjing Medical University&Jiangsu Cancer Hospital&Jiangsu Institute of Cancer ResearchNanjing 210009China

Spermatogenesis, maturation, capacitation and fertilization are precisely regulated by glycosylation. However, the relationship between altered glycosylation patterns and the onset and development of reproductive disorders is unclear, mainly limited by the lack of in situ imaging techniques for spermatozoa glycosylation. We developed an efficient and highly specific spermatozoa glycan imaging technique based on the robust chemoselective labeling of sialic acid(Sia) and N-acetyl-D-galactosamine(Gal/GalNAc). We further proposed a “tandem glycan chemoselective labeling” strategy to achieve simultaneous imaging of two types of glycans on spermatozoa. We applied the developed method to the spermatozoa from oligozoospermic patients and diabetic mice and found that these spermatozoa showed higher levels of Sia and Gal/Gal NAc expression than the normal groups. Moreover, spermatozoa from diabetic mice showed a severe decrease in number, viability, and forward motility, suggesting that in vivo glucose metabolism disorders may lead to an elevated level of spermatozoa glycosylation and have a correlation with the development of oligoasthenotspermia. Our work provides a research tool to reveal the relationship between glycosylation modification and spermatozoa quality, and a promising clue for the development of glycan-based reproductive markers.

关键词： spermatozoa Glycan Chemoselective labeling Oligoasthenotspermia Fluorescence imaging

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Participation of the inositol 1,4,5-trisphosphategated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa

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Asian Journal of Andrology 2020年第2期22卷 192-199页

作者： Ying-Ya Li Yan-Ping Jia Li-Yan Duan Kun-Ming Li Center of Reproductive Medicine Shanghai First Maternity and Infant HospitalTongji University School of MedicineShanghai 201204China

The acrosome reaction is a prerequisite for fertilization,and its signaling pathway has been investigated for decades.Regardless of the type of inducers present,the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium.Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers.In this study,we used a novel permeabilization tool BioPORTER?and first demonstrated its effectiveness in spermatozoa.The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER?and significantly reduced the calcium influx and acrosome reaction induced by progesterone,solubilized zona pellucida,and the calcium ionophore A23187.This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa.Moreover,we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions,which provides more reliable evidence for this process.In addition,by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER?in the presence or absence of calcium in the culture medium,we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx.This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

关键词： acrosome reaction human spermatozoa inositol 1,4,5-trisphosphate zona pellucida progesterone

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Taurine in semen extender modulates post–thaw semen quality,sperm kinematics and oxidative stress status in mithun(Bos frontalis)spermatozoa

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Asian pacific Journal of Reproduction 2022年第1期11卷 35-43页

作者： Perumal Ponraj Kobu Khate Kezhavituo Vupru ICAR-National Research Centre on Mithun MedziphemaNagaland–797106India ICAR-Central Island Agricultural Research Institute Port Blair744105Andaman and Nicobar IslandsIndia

Objective:To assess the effect of taurine on post-thaw semen quality parameters,sperm kinematics,antioxidant and oxidative stress profiles and sperm cholesterol efflux in mithun(Bos frontalis).Methods:A total of 50 ejaculates(n=25 samples)were selected based on biophysical parameters.Each sample was split into four equal aliquots after dilution with the Tris-citrate-glycerol extender.GroupⅠ,Ⅱ,ⅢandⅣcontained 0 mM(the control),25 mM,50 mM and 100 mM of taurine,respectively.Frozen-thawed samples were analysed for motility parameters(progressive forward and in bovine cervical mucus penetration test),kinetic and velocity parameters by computer-assisted sperm analyzer,viability,sperm and nuclear abnormalities,acrosome integrity,plasma membrane and nuclear integrities,sperm enzymatic leakage and biochemical(sperm cholesterol and oxidative stress)profiles.Results:The extender containing 50 mM taurine led to a significant enhancement in viability,acrosomal integrity,plasma membrane integrity,motility(progressive and in cervical mucus),and sperm cholesterol content and notably reduced sperm morphological and nuclear abnormalities,and leakage of intracellular enzymes compared to other taurine treated and untreated control groups(P<0.05).Moreover,in addition to significant improvement in kinetic and velocity profiles,50 mM taurine protected the integrity of acrosome and biochemical membranes than in the untreated control and other taurine treated groups.Inclusion of 50 mM taurine held a clear advantage over the control or 25 mM or 100 mM taurine in cryopreservation of mithun semen.Conclusions:Taurine(50 mM)supplementation in semen extender can be effectively utilized to reduce oxidative stress and improve post-thaw semen quality in mithun.

关键词： Antioxidants Cryopreservation Kinematic profiles Mithun Oxidative stress spermatozoa Taurine Semen quality Post-thaw

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Implementation of an in-house flow cytometric analysis of DNA fragmentation in spermatozoa

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Asian Journal of Andrology 2020年第3期22卷 246-251页

作者： Anne Sofie Rex Chunsen Wu Jorn Aagaard Jens Fedder Centre of Andrology and Fertility Clinic Odense University HospitalOdense 5000Denmark Aagaard Gynaecological Clinic Skejby 8200AarhusDenmark Research Unit of Gynaecology and Obstetrics Faculty of HealthUniversity of Southern DenmarkOdense 5000Denmark.

An increased amount of DNA fragme ntation in the spermatozoa(SDF)is linked to male in fertility.The Sperm Chromati n Structure Assay(SCSA)is widely used for analysis of SDF.However,the current software(SCSASoftR)linked to this assay is licensed and often located within larger diagnostic centers.In this study,we present a protocol for using other types of software than SCSASoftR to determine the SDF index(DFI)with clinical relevance.This protocol is engineered after collect!ng and analyzing 254 samples from fertility patients and sperm donors over a 15-month period.DFI is analyzed using a strict protocol where the spermatozoa are treated with a strong acid(pH 1.2)followed by acridine orange.DFI is determined by a standard flow cytometric software,FACSDiva 6.1.3.Analysis of the outcome of the fertility treatment is included for 137 patients receiving either intrauterine inseminations(IUI)or timed coitus(TC).The results show that the chance of pregnancy decli nes as DFI in creases.We also found that the male DFI affects the chanee of pregnancy independent of the female age.We have shown that a standard flow cytometric software can be used when determi ning a clinical releva nt DFI.These findings are a sign ificant step toward impleme nting the an alysis as a part of the routi ne,in・house diag no sing of the male fertility patient and subseque ntly optimizing the treatme nt course of the couple with reduced human and financial costs.

关键词： DNA fragme ntation flow cytometry in-house an alysis in trauteri ne in semi natio n Sperm Chromati n Structure Assay spermatozoa

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Proteomic analysis reveals dysregulated cell sign aling in ejaculated spermatozoa from in fertile men

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Asian Journal of Andrology 2019年第2期21卷 121-130页

作者： Luna Samanta Rakesh Sharma Zhihong Cui Ashok Agarwal American Center for Reproductive Medicine Cleveland ClinicClevelandOH 44195USA Department of Zoology School of Life SciencesRavenshaw UniversityCuttackOdisha 753003India Institute of Toxicology The Third Military Medical UniversityChongqing 400038China

Dysfunctional sperm maturation is the primary reason for the poor sperm motility and morphology in infertile men.spermatozoa from infertile men were fractioned on three-layer density gradient(80%,60%,and 40%).Fraction 1(Fl)refers to the least mature stage having the lowest den sity,whereas the fraction 4(F4)in eludes the most dense and morphologically mature motile spermatozoa.Fraction 2(F2)and fraction 3(F3)represent the intermediate stages.Proteins were extracted and separated by dimensional gel.Bands were digested with trypsin and analyzed on a LTQ-Orbitrap Elite hybrid mass spectrometer system.Functional annotations of proteins were obtained using bioinformatics tools and pathway databases.A total of 1585 proteins were detected in the four fractions of spermatozoa.A dysregulated protein turnover and protein folding may lead to accumulation of defective proteins or proteins that otherwise would have been eliminated during the process of maturation,resulting in the impairment of sperm fun ction.Aberra nt chaper one expressi on may be a major con tributing factor to the defective sperm function.Androge n receptor was predicted as a transcription regulator in one of the networks and the affected pathways were chaperone-mediated stress response,proteosomal pathway,and sperm function.The down regulation of key pathways and protei ns which compromises the fertilizi ng potential of spermatozoa may provide in sight into the mecha nisms that lead to male infertility.

关键词： androgen receptor chaperone immature sperm infertile men proteasome spermatozoa

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Antioxidant potential of pentoxifylline on spermatozoa of small ruminants

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Asian pacific Journal of Reproduction 2022年第3期11卷 138-145页

作者： Mazen Alomar Division of Animal Production Department of AgricultureAtomic Energy CommissionP.O.Box 6091DamascusSyria

Objective:To investigate antioxidant potential of pentoxifylline on spermatozoa of small ruminants including rams and bucks.Methods:The levels of hydrogen peroxide(H2O2)production in ram and buck spermatozoa incubated with 0(control),4 and 8 mM of pentoxifylline were measured after 45-min incubation.Then,the motility parameters of ram and buck spermatozoa incubated with 0(control),1 mM of H2O2,1 mM of H2O2 plus 4 mM of pentoxifylline,and 1 mM of H2O2 plus 8 mM of pentoxifylline were analysed.H2O2 was estimated using a fluorometric assay and spermatozoa motility characteristics were analyzed using computer aided sperm analyzer.Results:Pentoxifylline significantly decreased the levels of H2O2 produced from both ram and buck spermatozoa(Pspermatozoa were incubated with both H2O2 and two concentrations of pentoxifylline,the motility values rose significantly compared to those incubated with H2O2 alone(Pspermatozoa.Therefore,pentoxifylline could be recommended as antioxidant additive for spermatozoa of small ruminants under stress conditions.

关键词： Pentoxifylline spermatozoa Ram Buck Motiltiy Hydrogen peroxide H2O2 Antioxidants

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spermatozoa morphometry and ultrastructure in estuarine crocodile (Crocodylus porosus)

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Asian pacific Journal of Reproduction 2020年第2期9卷 104-108页

作者： Wan-Nor Fitri Haron Wahid Putra Tengku Rinalfi Dana Raj Yawah Donny Latip Qayyum Ab Aziz Abdul Malek Department of Veterinary Clinical Studies Faculty of Veterinary MedicineUniversiti Putra Malaysia43400 UPM SerdangSelangorMalaysia Research Centre for Wildlife Faculty of Veterinary MedicineUniversiti Putra Malaysia43400 UPM SerdangSelangorMalaysia Department of Veterinary Pre-Clinical Studies Faculty of Veterinary MedicineUniversiti Putra Malaysia43400 UPM SerdangSelangorMalaysia Sarang Buaya Pasir Gudang Aras 2 Menara AqabahJalan Bandar81700 Pasir GudangJohorMalaysia Wildlife Veterinary Section Ex-Situ Conservation DivisionDepartment of Wildlife and National Parks(PERHILITAN)Peninsular Malaysia

Objective: To evaluate normal spermatozoa morphometry and ultrastructure in estuarine crocodile (Crocodylus porosus). Methods: Four adult male crocodiles aged between 12-15 years, with an average snout to tail length of (3.15±0.01) m were selected for this study. Manipulation of the phallus digitally from the base of the penis was performed on four adult male crocodiles to facilitate the flow of semen to the sulcus. Semen was collected from all individuals for spermatozoa morphometry and ultrastructure study. Morphometry analysis was performed from eosin-nigrosin stained spermatozoa sample. Scanning electron microscopy was conducted to observe the surface ultrastructure of spermatozoa. Results: The morphology of crocodile spermatozoa was made up of acrosome, head, and tail which corresponded to (5.55±1.20) μm, (12.74±1.57) μm, and (70.67±4.40) μm, respectively. The total length of spermatozoa in estuarine crocodile was measured at (88.96±0.52) μm. The most common spermatozoa abnormalities found in the samples were cytoplasmic droplets. Conclusions: Normal morphology, morphometry of spermatozoa in estuarine crocodile which are vermiform shaped-head with a long tail along with questionably high cytoplasmic droplets count are described in this study.

关键词： spermatozoa Scanning electron microscopy Morphology Crocodile Cytoplasmic droplets

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Autophagy is increased in cryptorchid testis resulting in abnormal spermatozoa

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Asian Journal of Andrology 2019年第6期21卷 570-576页

作者： Marina G Yefimova Antoine Buschiazzo Agnes Burel Marie-Therese Lavault Celine Pimentel Guilhem Jouve Sylvie Jaillard Bernard Jegou Nicolas Bourmeyster Celia Ravel Univ Rennes CHU RennesLaboratoire de Biologie de la Reproduction-CECOSInsermEH ESPIrset(Institut de recherche en santeenvironnement et travail)-UMR_S 1085Rennes F-35000France Sechenov Institute of Evolutionary Physiology and Biochemistry Russian Academy of Sciences194223 St-PetersburgRussia Univ Rennes Biosit Platform-MRICRennes 35000France Univ Rennes CHU RennesDepartment of Gynecology Obstetric and Human ReproductionRennes 35000France Univ Rennes CHU RennesDepartment of CytogeneticIrset(Institute for Environmental and Occupational Health)-UMR_S 1085Rennes 35000France Univ Rennes InsermEH ESPIrset(Institute for Environmental and Occupational Health)-UMR_S 1085Rennes F-35000France University of Poitiers-CHU Poitiers STIM ERL 7003 CNRSPoitiers 86021France

Autophagy is invoIved in spermatogenesis by regulating germ cell maturation.This catabolic process increases with hyperthermic conditions to prevent the accumulation of damaged organelles.Cryptorchidism is associated with impairment of germ cell maturation revealed by the presence of immature forms of sperm cells in ejaculates.The aim of the present study was to evaluate the status of autophagy in sperm cells from cryptorchid patients.Semen samples of cryptorchid patients and normozoospermic controls were analyzed by immuno cytochemistry and electron microscopy.Autophagy protei ns,autophagy-related protein 9(ATG9)and microtubule-associated protein,lA/lB-light chain 3(LC3)were localized by immunocytochemistry on the acrosome and on the equatorial segment of sperm cells.LC3 was also detected in the midpiece of cryptorchid sperm tail.Autophagy substrate p62 protein was present in the acrosome and in the postequatorial segment of sperm in control samples,but not in the cryptorchid ones.Transmission electron microscopy revealed double-membrane-limited autophagosomes in postequatorial part of spermatozoa head and midpiece in cryptorchid samples.Partly degraded mitochondria were frequently discerned in autophagic vacuoles.In conclusion,autophagy is increased in sperm cells from patients with cryptorchid history comparatively to control.Our work provides insights into the role of autophagy in the maturation and survival of human male gametes in pathological conditions.Thus,regulating autophagy could represent a potential way to improve sperm quality in cryptorchid men.

关键词： autophagy cryptorchidism mitophagy spermatozoa

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Interaction between basigin and monocarboxylate transporter 2 in the mouse testes and spermatozoa

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Asian Journal of Andrology 2016年第4期18卷 600-606页

作者： Cheng Chen Mamiko Maekawa Kenji Yamatoya Masami Nozaki Chizuru Ito Toshihiko Iwanaga Kiyotaka Toshimori Department of Anatomy and Developmental Biology Graduate School of Medicine Chiba University Chiba Japan Department of Cell Biology Research Institute for Microbia Diseases Osaka University Osaka Japan Laboratory of Histology and Cytology Department of Anatomy Graduate School of Medicine Hokkaido University Hokkaido Japan

Basigin is a member of the immunoglobulin superfamily and plays various important roles in biological events including spermatogenesis. To examine the basigin molecular variants during spermatogenesis and sperm maturation in the mouse, immunoprecipitated basigin samples from testis and epididymal spermatozoa were analyzed by liquid chromatography-mass spectrometry/mass spectrometry （LC-MS/MS）. The results demonstrated that basigin molecules from the testis and spermatozoa were separable into two major bands and that the differences in the molecular sizes were possibly because of an endoproteolytic cleavage. Since basigin is known to be a chaperone for the monocarboxylate transporter 1 （MCT1）, the localization of basigin, MCT1 and MCT2 was examined during postnatal testicular development. Immunohistochemical studies showed different expression patterns of MCT1 and MCT2. MCT1 was localized on the surface of spermatogonia, spermatocytes, and spermatids. In contrast, MCT2 appeared on the principal piece of spermatozoa in the testis, where basigin was also observed. In mature epididymal spermatozoa, MCT2 was located on the midpiece, where basigin co-localized with MCT2 but not with MCT1. Furthermore, MCT2 was immunoprecipitated with basigin in mouse testes and sperm. These results suggest that basigin has a functional role as a binding partner with MCT2 in testicular and epididymal spermatozoa.

关键词： basigin monocarboxylate transporter sperm maturation spermatogenesis spermatozoa testis

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Altered microRNA expression profiles of human spermatozoa in normal fertile men of different ages

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Asian Journal of Andrology 2023年第6期25卷 737-744页

作者： Ming-Jia Zhao Yao-Nan Zhang Yong-Ping Zhao Xian-Bing Chen Bao-Sheng Han Ning Ding Yi-Qun Gu Shu-Song Wang Jing Ma Mei-Ling Liu Graduate School of Peking Union Medical College Peking Union Medical CollegeChinese Academy of Medical SciencesBeijing 100730China National Health Commission Key Laboratory of Male Reproductive Health National Research Institute for Family PlanningBeijing 100081China Department of Reproduction and Genetics Maternity and Child Health Care HospitalTangshan 063000China Department of Family Planning and Reproductive Medicine Peking University People’s HospitalBeijing 100081China Hebei Key Laboratory of Reproductive Medicine Hebei Institute of Reproductive Health Science and TechnologyShijiazhuang 050071China

MicroRNAs(miRNAs)are mediators of the aging process.The purpose of this work was to analyze the miRNA expression profiles of spermatozoa from men of different ages with normal fertility.Twenty-seven donors were divided into three groups by age(Group A,n=8,age:20–30 years;Group B,n=10,age:31–40 years;and Group C,n=9,age:41–55 years)for high-throughput sequencing analysis.Samples from 65 individuals(22,22,and 21 in Groups A,B,and C,respectively)were used for validation by quantitative real-time polymerase chain reaction(qRT-PCR).A total of 2160 miRNAs were detected:1223 were known,937 were newly discovered and unnamed,of which 191 were expressed in all donors.A total of 7,5,and 17 differentially expressed microRNAs(DEMs)were found in Group A vs B,Group B vs C,and Group A vs C comparisons,respectively.Twenty-two miRNAs were statistically correlated with age.Twelve miRNAs were identified as age-associated miRNAs,including hsa-miR-127-3p,mmu-miR-5100_L+2R-1,efu-miR-9226_L-2_1ss22GA,cgr-miR-1260_L+1,hsa-miR-652-3p_R+1,pal-miR-9993a-3p_L+2R-1,hsa-miR-7977_1ss6AG,hsa-miR-106b-3p_R-1,hsa-miR-186-5p,PC-3p-59611_111,hsa-miR-93-3p_R+1,and aeca-mir-8986ap5_1ss1GA.There were 9165 target genes of age-associated miRNAs.Gene Ontology(GO)analysis of the target genes identified revealed enrichment of protein binding,membrane,cell cycle,and so on.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of age-related miRNAs for target genes revealed 139 enriched pathways,such as signaling pathways regulating stem cell pluripotency,metabolic pathways,and the Hippo signaling pathway.This suggests that miRNAs play a key role in male fertility changes with increasing age and provides new evidence for the study of the mechanism of age-related male fertility decline.

关键词： age fertility microRNA spermatozoa

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