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AMP-activated kinase in human spermatozoa： identification, intracellular localization, and key function in the reRulation of sperm motility

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Asian Journal of Andrology 2017年第6期19卷 707-714页

作者： Violeta Calle-Guisado Ana Hurtado de Llera David Martin-Hidalgo Jose Mijares Maria C Gil Ignacio S Alvarez Maria J Bragado Luis J Garcia-Marin Research Group of Intracellular Signalling and Technology of Reproduction (SINTREP) School of Veterinary Medicine University of Extremadura Caceres Spain Assisted Reproduction Unit at the-Minimally Invasive surgery Center Jesos Uson (CCMIJU) Caceres Spain Norba Clinic Caceres Spain Research Group of Reproduction and Embryo Development (REDES) University of Extremadura Badajoz Spain Extremadura Institute of Assisted Reproduction (IERA) Badajoz Spain.

AMP-activated kinase （AMPK）, a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work＇s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. human spermatozoa motility was evaluated by an Integrated Semen Analysis System （ISAS） in the presence or absence of the AMPK inhibitor compound C （CC）. AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed： percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

关键词： AMP-activated kinase human spermatozoa immunolocalization sperm motility sperm quality

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Estimate of oxygen consumption and intracellular zinc concentration of human spermatozoa in relation to motility

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Asian Journal of Andrology 2003年第1期5卷 3-8页

作者： Ralf R.Henkel Kerstin Defosse Hans-Wilhelm Koyro Norbert Weissmann Wolf-Bernhard Schill Center for Dermatology and Andrology University D-35385 Giessen GermanyCenter for Dermatology and Andrology University D-35385 Giessen GermanyInstitute for Plant Ecology University D-35385 Giessen GermanyCenter for Internal Medicine Justus Liebig University D-35385 Giessen GermanyCenter for Dermatology and Andrology University D-35385 Giessen Germany

Aim: To investigate the human sperm oxygen/energy consumption and zinc content in relation to motility. Methods: In washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility. Results: The oxygen consumption averaged 0.24μmol/106 sperm×24 h, 0.28μmol/106 live sperm×24 h and 0.85μmol/106 live & motile sperm×24 h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/106 motile spermatozoa×24 h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/106 spermatozoa×24 h. The correlation of the oxygen/ energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, respectively). However, when correlating sperm energy consumption with the zinc content, a significant positive relation (r=0.323; P=0.01) was observed. Conclusion: Poorly motile sperm are actually wasting the available energy. Moreover, our data clearly support the 'Geometric Clutch Model' of the axoneme function and demonstrate the importance of the outer dense fibers for the generation of sperm motility, especially progressive motility.

关键词： oxygen energy consumption human spermatozoa sperm motility zinc

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Fluorescent probes for the detection of reactive oxygen species in human spermatozoa

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Asian Journal of Andrology 2020年第5期22卷 465-471页

作者： Sara Escada-Rebelo Francisca G Mora Ana P Sousa Teresa Almeida-Santos Artur Paiva Joao Ramalho-Santos phD Programme in Experimental Biology and Biomedicine CNC-Center for Neuroscience and Cell BiologyUniversity of CoimbraCoimbra 3004-504Portugal Biology of Reproduction and Stem Cell Group CNC-Center for Neuroscience and Cell BiologyUniversity of CoimbraCoimbra 3004-504Portugal IIIUC-Institute for Interdisciplinary Research Casa Costa AlemaoUniversity of CoimbraCoimbra 3030-789Portugal Reproductive Medicine Unit University Hospitals of CoimbraCoimbra 3004-561Portugal Faculty of Medicine University of CoimbraCoimbra 3000-370Portugal Clinical Pathology Unit University Hospitals of CoimbraCoimbra 3004-561Portugal Department of Life Sciences University of CoimbraCoimbra 3000-456Portugal

Reactive oxygen species(ROS)production is a by-product of mitochondrial activity and is necessary for the acquisition of the capacitated state,a requirement for functional ***,an increase in oxidative stress,due to an abnormal production of ROS,has been shown to be related to loss of sperm function,highlighting the importance of an accurate detection of sperm ROS,given the specific nature of this *** this work,we tested a variety of commercially available fluorescent probes to detect ROS and reactive nitrogen species(RNS)in human sperm,to define their *** both flow cytometry(FC)and fluorescence microscopy(FM),we confirmed that MitoSOX™Red and dihydroethidium(DHE)detect superoxide anion(as determined using antimycin A as a positive control),while DAF-2A detects reactive nitrogen species(namely,nitric oxide).For the first time,we also report that RedoxSensor™Red CC-1,CellROX®Orange Reagent,and MitoPYl seem to be mostly sensitive to hydrogen peroxide,but not ***,mean fluorescence intensity(and not percentage of labeled cells)is the main parameter that can be reproducibly monitored using this type of methodology.

关键词： flow cytometry fluorescent probes human spermatozoa oxidative stress reactive oxygen species

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Adriamycin induces H2AX phosphorylation in human spermatozoa

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Asian Journal of Andrology 2008年第5期10卷 749-757页

作者： Zhong-Xiang Li Ting-Ting Wang Yan-Ting Wu Chen-Ming Xu Min-Yue Dong Jian-Zhong Sheng He-Feng Huang Department of Reproductive Endocrinology Women＇s Hospital Zhejiang University School of Medicine Hangzhou 310006 China Department of Pharmacology and Therapeutics Faculty of Medicine University of Calgary Calgary Alberta T2N 4N1 Canada

Aim： To investigate whether adriamycin induces DNA damage and the formation of γH2AX （the phosphorylated form of histone H2AX） foci in mature spermatozoa. Methods： human spermatozoa were treated with adriamycin at different concentrations, γH2AX was analyzed by immunofluorescent staining and flow cytometry and doublestrand breaks （DSB） were detected by the comet assay. Results： The neutral comet assay revealed that the treatment with adriamycin at 2 μg/mL for different times （0.5, 2, 8 and 24 h）, or for 8 h at different concentrations （0,4, 2 and 10 μg/mL）, induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of γH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP 1 with γH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase （PI3K） family, abolished the co-appearance of these two proteins with γH2AX. Conclusion： human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/ repair proteins as somatic cells.

关键词： adriamycin human spermatozoa DNA double strand-breaks γH2AX

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Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa

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Asian Journal of Andrology 2008年第6期10卷 829-836页

作者： Jacqueline Leβig Uta Reibetanz Jiirgen Arnhold Hans-Jtirgen Glander Institute for Medical Physics and Biophysics Medical Faculty University of Leipzig Leipzig D-04107 Germany Department of Dermatology/Andrological Training Centre of the European Academy of Andrology University of Leipzig Leipzig D-04103 Germany

Aim： To determine the cellular distribution of secretory phospholipase A2 （sPLA2） in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods： Acrosome reaction of spermatozoa was triggered by calcimycin. human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA2 and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Results： Although sPLA2 was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA2 was associated with enhanced binding of annexin V-fluoroscein isothiocyanate （FITC） to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA2, disturbance of acrosome structure, and loss of vitality. Conclusion： The ability of spermatozoa to release secretory phospholipase A2 is related to the acrosomal state. Premature destabi- lization of the acrosome and loss of sPLA2 can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA2 in intact spermatozoa might be an additional parameter for evaluating sperm quality.

关键词： acrosome reaction elastase human spermatozoa inflammation secretory phospholipase A2

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Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

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Asian Journal of Andrology 2006年第3期8卷 273-279,385页

作者： BabyAhmedAli Halima-HassanSalem Research Center for Reproductive Medicine Cell Biology and Medical Genetics DepartmentShantou University Medical CollegeShantou 515041China Nucleic Acid Research DepartmentGenetic Engineering and Biotechnology Research InstituteMubarak City for Scientific Research and Technology ApplicationsNew Borg El-Arab City 21934Alexandria.Egypt

Aim:To detect the expression of hepatitis B virus(HBV)genes(HB S and C genes)in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization(IVF) ***:human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF *** chain reaction(PCR)was used to detect HB S and pre-Core/Core(pre-C/C)coding genes both in one-and two-cell *** transcription-PCR(RT-PCR)analysis was used to study the expression of the two *** in situ hybridization(FISH)analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic ***:Both HB S and pre-C/C coding genes are present and transcribed in one-and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA ***:Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic *** results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

关键词： hepatitis B virus gene expression hamster ovary human spermatozoa in vitro fertilization

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Selection and Evaluation of Optimal Reference Genes for Quantitative Reverse Transcription-Polymerase Chain Reaction Analyses of Gene Expression in human spermatozoa

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Reproductive and Developmental Medicine 2020年第4期4卷 212-218页

作者： Luo Chun-Hai Tang Yun-Ge Hong Shi-Hao Tang Yuan Zhang Ying Sun Fei Institute of Reproductive Medicine School of MedicineNantong UniversityNantong 226001China Key Laboratory of Male Reproductive and Genetics National Health and Family Planning CommissionGuangzhou 510060China

Objective:Optimal reference genes are critical for accurate normalization and reliable interpretation of gene expression quantification ***,several strategies have been utilized for validating reference genes in different human ***,no universal reference genes have been described that accurately summarize transcriptional activity in human ***:Using quantitative reverse transcription-polymerase chain reaction(RT-qPCR),we evaluated ten commonly used candidate reference genes between two groups of human cryopreserved donor sperm with different pregnancy *** assessed the stability of reference genes using three different algorithms,namely geNorm,NormFinder,and *** then identified the most stable reference ***:Male-enhanced antigen 1(MEA1)was identified as the most stably expressed reference gene,followed by testis-enhanced gene transcript(TEGT).Conclusions:We comprehensively identified MEA1 and TEGT as the most stably expressed reference genes for the normalization of gene expression data in human spermatozoa.

关键词： human spermatozoa Quantitative Reverse Transcription-Polymerase Chain Reaction Reference Gene

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The expression of vitronectin,a ligand of integrin,on human spermatozoa and its role in fertilization

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Asian Journal of Andrology 2001年第1期3卷 74-74页

作者： Bronson Rictard State University of New York at Story Brook USA

Objective: To study the vitronectin expression in human spermato-zoa and its role in fertilization. Methods: spermatozoa from 14 fer-tile and 8 infertile men with normal semen data were studied. Follow-ing recovery of motile populations by swim-up, spermatozoa were ca-pacitated and immunostained with rabbit anti-human Vn polyclonal an-tibody and goat anti-rabbit IgG-FTTC second antibody. The percentageof spermatozoa expressing Vn was determined using a FAScan flowcytometer. Meanwhile, the fertilizing ability of capacitated spermato-zoa was determined with human spermatozoa zona-free hamster eggpenetration assay (SPA). Results: The mean ± s proportion ofspermatozoa expressing Vn of fertile men was 21.24% ± 11.70% and3.64±3.27% for infertile men (P 10%, but that in the infertile group ,human capacitated spermatozoaand its correlation with the fertilizing ability. The abnormal expressionof Vn on human spermatozoa may be one of the unexplained infertilereasons. (Reprod Contracep 2001; 21: 24-28)

关键词： vitronectin (Vn) human spermatozoa fertilization

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Analysis of the Changes of Movement Function and Viability in human spermatozoa Induced by Reactive Oxygen Species

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Asian Journal of Andrology 2003年第4期 300-300页

作者： Xue-Jun Shang, Zhang-Qun Ye, Cheng-Liang Xiong, Dong-Hui Huang, Yong-Mei WangDepartment of Urology, Tongji Hospital, Institute of Family Planning Research, Tongji Medical College, Huazhong Universityof Science and Technology, Wuhan 430030, ChinaLaboratory of Reproduction & Genetics, Nanjing GeneralHospital of Nanjing Command, PLA, Nanjing 210002, China

Objective: To study the influence of reactive oxygen species (ROS) on the mobility and viability of human spermatozoa. Methods: spermatozoa with normal function were selected from human semen samples by the Percoll gradient centrifugation technique. ROS were generated by the hypoxanthine-xanthine oxidase system and were incubated with the spermatozoa under aerobic environment. Movement parameters of spermatozoa were analyzed by the computer-assisted semen analysis (CASA) system. Results: Thirty min after incubation with ROS, the motility, curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) of the spermatozoa were significantly decreased (P0.05). After 60 min incubation, the motility was almost lost with all the movement parameters close to zero. Conclusion: When normal spermatozoa were incubated with ROS, the mobility was decreased. It is believed that ROS may be one of t

关键词： reactive oxygen species human spermatozoa mobility viability

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Protein tyrosine phosphorylation of the human sperm head during capacitation： immunolocalization and relationship with acquisition of sperm-fertilizing ability

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Asian Journal of Andrology 2010年第6期12卷 853-861页

作者： Arcangelo Barbonetti Maria Rosaria C. Vassallo Giuliana Cordeschi Dimitrios Venetis Andrea Carboni Alessandra Sperandio Giorgio Felzani Sandro Francavilla Felice Francavilla Andrology Unit Department of Internal Medicine University of L＇Aquila Coppito 67100 L＇Aquila Italy Istituto di Ricovero e Cura a Carattere Scientifico S.Raffaele Sulmona 67039 Italy

The occurrence of tyrosine phosphorylation （TP） in the sperm head during capacitation has been poorly investigated, and no data exist on the relationship of its dynamics with the acquisition of sperm fertilizing ability. This study localized TP of head proteins in human spermatozoa during capacitation and explored its relationship with acquisition of the ability to display progesterone （P）-stimulated acrosome reactions （ARs） and to penetrate zona-free hamster oocytes. By immunofluorescence, TP immunoreactivity was revealed in the acrosomal region of formaldehyde-fixed/unpermeabilized samples, whereas it was abolished in fixed/permeabilized samples, in which TP immunoreactivity was high in the principal piece. No TP immunoreaetivity was detectable in unfixed spermatozoa. Head TP immunoreactivity was localized externally to the acrosome, close to the cytoplasmic membrane, as assessed by transmission electron microscopy. The increase in head TP was an early event during capacitation, occurring within 1 h in capacitating conditions. At this time, the P-stimulated ARs were also increased, whereas egg penetration was as poor as in uncapacitated spermatozoa. At 5 h of capacitation, the extent of neither head TP nor the P-induced ARs were greater than that at 1 h, whereas egg penetration had significantly increased. Seminal plasma inhibited head TP, P-induced ARs and egg penetration. None of these inhibitory effects, unlike those on tail TP, were prevented by the cAMP analogue dbcAMP （N,2-O-dibutyryladenosine 3＇,5＇-cyclic monophosphate）. In conclusion, head TP is a subsurface event occurring early during capacitation and is closely related to acquisition of the ability to display P-stimulated ARs, whereas the ability to fuse with oolemma and to decondense is a later capacitation-related event.

关键词： acrosome reaction capacitation human spermatozoa sperm-oocyte fusion tyrosine phosphorylation

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