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

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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Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

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Asian Journal of Andrology 2005年第1期7卷 86-92,页

作者： ShyamS.R.Allamaneni AshokAgarwal SreedharRama PavithraRanganathan RakeshK.Sharma Center for Advanced Research in Human Reproduction Infertility and Sexual Function Glickman Urological Institute and Department of Obstetrics-Gynecology Cleveland Clinic Foundation Cleveland Ohio USA

Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. Methods: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10~6 vs. 17.6 x 10~6), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Conclusion: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART. i

关键词： spermatozoa cryopreservation density gradient centrifugation sperm motility

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18,X,Y aneuploidies and transmission electron microscopy studies in spermatozoa from five carriers of different reciprocal translocations

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Asian Journal of Andrology 2009年第3期11卷 325-332页

作者： Elena Moretti Nicola Antonio Pascarelli Valentina Giannerini Michela Geminiani Cecilia Anichini Giulia Collodel Department of Biomedical Sciences Applied Biology Section University of Siena Siena 53100 Italy Department of Pediatrics Obstetrics and Reproductive Medicine University of Siena Siena 53100 Italy

We analysed ejaculated spermatozoa from five infertile men with different balanced reciprocal translocations to contribute to the study of meiotic segregation of chromosomes 18, X and Y and also to evaluate sperm morphology by transmission electron microscopy （TEM） analysis. Conventional lymphocyte karyotype analyses highlighted dif- ferent reciprocal balanced translocations： t（12; 13）, t（4;9）, t（X;8）, t（8; 10） and t（3; 16）. Semen analysis was performed by light and TEM. Fluorescence in situ hybridization was performed directly on sperm nuclei using centromeric probes for chromosomes 18, X and Y. The carriers of the balanced reciprocal translocations considered in the present study showed a very similar pattern of sperm pathologies： diffused presence of apoptosis and immaturity. All patients showed meiotic segregation derangements, highlighted by the presence of sperm diploidies and sex chromosome disomies particularly related to the failure of the first meiotic division. However, an increased incidence of chromosome 18 aneuploidy was detected in spermatozoa from t（X;8） and t（8;10） carriers. We have also reported values from sex chromosomes such as t（X;8）, although the X chromosome was involved in translocation. Since patients with reciprocal translocations and spermatogenetic impairment are candidates for intracytoplasmic sperm injection cycles, the study of sperm parameters, and particularly of the level of aneuploidy rates, would provide better information for couples at risk and would contribute to the data in the literature for a better understanding of the effects of chromosomal rearrangement on the whole meiotic process and, in particular, on chromosomes not involved in translocation.

关键词： electron microscopy fluorescence in situ hybridization altered karyotype spermatozoa reciprocal translocation

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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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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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Proteomic changes in mammalian spermatozoa during epididymal maturation

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Asian Journal of Andrology 2007年第4期9卷 554-564页

作者： R. John Aitken Brett Nixon Minjie Lin Adam J. Koppers Yun H. Lee Mark A. Baker The ARC Centre of Excellence in Biotechnology and Development University of Newcastle Newcastle NSW 2308Australia Discipline of Biological Sciences University of Newcastle Newcastle NSW 2308 Australia

Epididymal maturation is associated with the activation of a cAMP-induced tyrosine phosphorylation cascade, which is ultimately associated with the expression of capacitation-dependent sperm functions, such as hyperactivated movement and acrosomal exocytosis. As spermatozoa progress through the epididymis they first acquire the capacity to phosphorylate tyrosine on targets on the principal piece, followed by the midpiece. By the time these cells have reached the cauda epididymidis they can phosphorylate the entire tail from neck to endpiece. This particular pattern of phosphorylation is associated with the ontogeny of fully functional spermatozoa that are capable of fertilizing the oocyte. Proteomic analyses indicate that this change is associated with the phosphorylation of several mitochondrial proteins, creation of a mitochondrial membrane potential and activation of mitochondrial free radical generation. At least in rodent species, activation of sperm mitochondria appears to be a particularly important part of epididymal maturation. （Asian J Androl 2007 July; 9： 554-564）

关键词： epididymis mitochondria spermatozoa tyrosine phosphorylation

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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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Apoptosis and DNA damage in human spermatozoa

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Asian Journal of Andrology 2011年第1期13卷 36-42页

作者： R John Aitken Adam J Koppers ARC Centre of Excellence in Biotechnology and Development Priority Research Centre in Reproductive Science Discipline of Biological Sciences School of Environmental and Life Sciences University of Newcastle Callaghan NSW 2308 Australia Department of Anatomy and Developmental Biology School of Biomedical Sciences Monash University Clayton VIC 3800 Australia

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apeptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

关键词： apoptosis DNA damage oxidative stress spermatozoa

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Whither must spermatozoa wander？ The future of laboratory seminology

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Asian Journal of Andrology 2010年第1期12卷 99-103页

作者： R. JohnAitken ARC Centre of Excellence in Biotechnology and Development Discipline of Biological Sciences University of NewcastleCallaghan NSW 2308 Australia

This commentary celebrates the publication of the 5th for the Examination and Processing of Human Semen edition of the World Health Organization Laboratory Manual This is the most complete text to date on the creation of a conventional semen profile and includes invaluable reference limits for specific aspects of semen quality based on the analysis of over 1 900 recent fathers. The new edition of the manual also includes detailed protocols for monitoring different aspects of sperm function and new chapters on the preparation of spermatozoa for assisted conception and cryopreservation. Given that this publication is the definitive statement on how to perform a descriptive semen analysis, we might speculate on the future of this field and the sorts of tests that might feature in future editions of the manual. Cell biologists are currently being empowered by the ＇omics revolution, which is placing at their disposal technologies of unprecedented power to examine the biochemical composition of cells such as spermatozoa. Indeed, spermatozoa are perfect vehicles for this kind of analysis because they can be obtained as extremely pure suspensions, exist naturally in isolation and can be induced to express their capacity for fertilization and the initiation of embryonic development in vitro. The application of ＇omics technologies to these cells, in concert with detailed assessments of their functional competence, should provide insights into the biochemical basis of defective semen quality. This information will then help us understand the causes of male infertility and to develop rational methods for its treatment and possible prevention.

关键词： assisted conception DNA damage male infertility miscarriage semen seminology spermatozoa

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Antioxidative effect of fullerenol on goat epididymal spermatozoa

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Asian Journal of Andrology 2002年第2期4卷 149-152页

作者： M.ArulMurugan BinduGangadharan 等 SchoolofLifeSciences PondicherryUniversityPondicherry605014India

OBJECTIVE:To evaluate the effect of fullerenol on the antioxidant system of goat epididymal sperm.;METHODS:Fresh epididymides of adult goats were obtained from local slaughter houses and sperm were collected by chopping the epididymis in modified Ringer's phosphate solution (RPS medium). After several washings the sperm samples were equally dispersed in RPS medium and incubated with fullerenol (1, 10 and 100 micromol) and FeSO(4)/ascorbate (40/200 micromol) with or without fullerenol (1, 10 and 100 micromol) for 3 h at 32 degree C. After incubation, an aliquot of sperm samples were homogenized and centrifuged and the supernatant used for biochemical studies.;RESULTS:In FeSO(4)/ascorbate-incubated samples, the activities of antioxidant enzymes, superoxide dismutase, glutathione peroxidase and glutathione reductase, were decreased while lipid peroxidation increased as compared to the control sperm samples. In fullerenol-incubated sperm samples, the activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were increased while lipid peroxidation was decreased in a dose-dependent manner. Co-incubation of sperm with fullerenol (1,10 and 100 micromol) and FeSO(4)/ascorbate (40/200 micromol) increased the activities of antioxidant enzymes and prevented the iron-induced elevation of lipid peroxidation in a dose-dependent manner.;CONCLUSIONS:Fullerenol reduces iron-induced oxidative stress in epididymal sperm of goat by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation.

关键词： Antioxidant enzymes Fullerenol Goat Iron Lipid peroxidation Oxidative stress spermatozoa

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