Sperm Proteome Maturation in the Mouse Epididymis

In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.     

Ejaculated Mouse Sperm Enter Cumulus-Oocyte Complexes More Efficiently In Vitro than Epididymal Sperm

The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca²⁺ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca²⁺ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo.     

经皮附睾穿刺取精术在男性不育症诊疗中的应用(附58例报告)

目的:探讨经皮附睾穿刺取精术在男性梗阻性无精子症患者的不育诊治中的应用价值.方法:对58例临床诊断为无精子症的患者,用模型法测量睾丸体积,化学发光法测定血清性激素水平,用7号蝶形针头穿刺附睾头,同时抽吸附睾液.结果:58例无精子症患者中,31例附睾液中可见精子,其中睾丸体积正常者为27例,睾丸体积偏小者为4例;血清FSH水平正常者为28例,血清FSH水平增高者为3例.27例未见精子者,其中睾丸体积正常者为15例,睾丸体积偏小者为12例;血清FSH正常者为17例,血清FSH增高者为10例.结果显示睾丸体积正常的患者,穿刺成功率明显高于睾丸体积偏小者,差异有显著性(P<0.05);血清FSH水平正常的患者,穿刺成功率明显高于FSH水平增高者,差异有显著性(P<0.05).结论:经皮附睾穿刺取精术可简便、快速地鉴别梗阻性和非梗阻性无精子症,也是严重不育症患者获取精子的理想方法.     

Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

低温保护剂加入/取出过程中细胞体积的极值

低温保护剂是细胞低温保存过程中必不可少的,其加入／取出过程受到细胞体积变形极限的限制．在两参数细胞渗透模型的基础上,得到了细胞在低温保护剂加入／取出过程中细胞内水体积和细胞体积极值的解析解,分析了低温保护剂的渗透性和初始浓度差对细胞体积极值的影响。     

Doxycycline Alters the Porcine Renal Proteome and Degradome during Hypothermic Machine Perfusion

Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

The Rhesus Macaque (Macaca mulatta) Sperm Proteome

Mass spectrometry based proteomics has facilitated sperm composition studies in several mammalian species but no studies have been undertaken in non-human primate species. Here we report the analysis of the 1247 proteins that comprise the Rhesus macaque (Macaca mulatta) sperm proteome (termed the MacSP). Comparative analysis with previously characterized mouse and human sperm proteomes reveals substantial levels of orthology (47% and 40% respectively) and widespread overlap of functional categories based on Gene Ontology analyses. Approximately 10% of macaque sperm genes (113/1247) are significantly under-expressed in the testis as compared with other tissues, which may reflect proteins specifically acquired during epididymal maturation. Phylogenetic and genomic analyses of three MacSP ADAMs (A-Disintegrin and Metalloprotease proteins), ADAM18-, 20- and 21-like, provides empirical support for sperm genes functioning in non-human primate taxa which have been subsequently lost in the lineages leading to humans. The MacSP contains proteasome proteins of the 20S core subunit, the 19S proteasome activator complex and an alternate proteasome activator PA200, raising the possibility that proteasome activity is present in mature sperm. Robust empirical characterization of the Rhesus sperm proteome should greatly expand the possibility for targeted molecular studies of spermatogenesis and fertilization in a commonly used model species for human infertility.The application of mass spectrometry (MS) based proteomics, coupled with whole genome annotation of an increasing number of species, has greatly extended our knowledge of sperm composition. Traditional methods used to assess sperm composition, including the use of sperm-specific antibodies and 2D gel electrophoresis, have identified a limited number of sperm proteins. These traditional studies have been augmented in recent years by the use of high throughput and highly sensitive MS (shotgun proteomics) that have substantially increased the accuracy of peptide identification, resulting in a significant increase in proteome coverage. Indeed, advances in MS instrumentation, data acquisition, and the availability of genome annotations have, for example, increased sperm proteome coverage in Drosophila from 381 (1) to 1108 proteins (2) over a five year period.Two main MS based methodologies have been applied to study sperm composition, including (i) 2D PAGE followed by spot excision and MS and (ii) digestion of proteins, followed by MS/MS analysis of the resulting peptides (3). Although each method has its own advantages and disadvantages, a far greater level of proteome coverage is obtained using MS/MS (4). A previous comparative study found that each method identified proteins not found in the other and vice versa, and therefore it has been suggested that these methods should be used to complement each other (5). Thus, although no single methodology yet exists capable of producing a complete whole cell proteome, MS/MS methods provide deeper and broader coverage and are therefore the current method of choice. Shotgun proteomics has characterized sperm proteomes in a variety of taxa including plants, invertebrates and mammals such as human, mouse, rat, and bull (3, 6–11). These studies achieve varying levels of proteome coverage as a result of several factors including the choice of MS equipment, sample acquisition, purification, solublization, and fractionation schemes. Although these different approaches make direct comparisons difficult they nevertheless have provided invaluable information regarding the composition of sperm and have helped to identify novel proteins that play important roles in sperm function and reproduction.In this study we use MS based proteomics to elucidate the sperm proteome of a species of old world monkey, the Rhesus macaque (Macaca mulatta). Due primarily to their genetic and physiological similarities to humans, Rhesus macaques are the most widely used nonhuman primate model system for basic and applied biomedical research (12). Rhesus macaques are also used extensively as a model of human reproduction where numerous similarities at the molecular level have been observed between gametes of the two species, and why Rhesus macaques have become a useful model system for fertility and assisted reproductive technology research (13). A more complete knowledge of the sperm proteome will facilitate reproductive studies using the Rhesus macaque as a model organism. However, despite its widespread use in reproductive biology, the macaque sperm proteome (MacSP)¹ has yet to be characterized.Although insight into the MacSP will facilitate reproductive studies using the Rhesus macaque as a model organism, this knowledge can also be used to better understand the composition of human sperm. Sperm mature and gain fertilization competency as they traverse the epididymis, a specialized duct that connects the testis to the vas deferens (14). During the maturation process, sperm lose or modify a number of their surface proteins and gain additional transient or permanent surface proteins in a well-organized manner, and it is only after emerging from the cauda epididymis that sperm are motile and considered fertilization competent (14, 15).Proteomic studies of human sperm have been undertaken (3, 6, 10), identifying between 98–1760 sperm proteins, however these studies used sperm from ejaculates which complicates sperm proteome analysis. A previous study identified 923 proteins present in human seminal plasma (16), which is likely to be only a fraction of the seminal plasma proteome. Human sperm proteome data sets derived from human ejaculates makes it difficult to differentiate which of the identified proteins are sperm or seminal plasma constituents. For example, a major seminal protein family, the semenogelins are not expressed in the testis but are found in sperm proteomes determined from ejaculates (6, 10). Such highly abundant seminal proteins may mask lower abundance integral sperm proteins and inhibit their identification by MS. In order to avoid these problems, we collected mature sperm directly from the cauda epididymis of the Rhesus macaque, thus avoiding contamination from seminal plasma proteins.In the present study, sperm proteins were separated using 1D SDS-PAGE, digested and the resulting peptides analyzed by LC MS/MS. Using high stringency parameters for peptide identification, we conservatively identified 1247 proteins from purified samples of Rhesus macaque sperm. Given their close evolutionary relationship, the Rhesus macaque and human share 93% nucleotide homology (12). Data from this study can be used to complement what is currently known about the composition of human sperm and provides a more useful proxy of human sperm proteome composition than the proteomes of other non-primate mammals for which data is available. Studies of sperm composition, especially those in human, can be applied to develop novel molecular based clinical diagnostic tests of sperm quality, which is currently limited to evaluating parameters such as sperm count, morphology and motility. In addition, knowledge of sperm components can lead to the discovery of novel contraceptives and infertility treatments.     

Seminal Plasma Proteome as an Indicator of Sperm Dysfunction and Low Sperm Motility in Chickens

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Highlights

• •Quantitative proteomes of chicken seminal plasma associated with sperm motility.
• •High abundant acrosome and mitochondrial proteins were noted in LSM seminal plasma.
• •Decreased total antioxidant capacity was highlighted in seminal plasma of LSM.
• •Lack of membrane, acrosome and mitochondrial integrity and high ROS may induce LSM.

     

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

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Highlights

• •Matrisome content significantly changes with development and perlecan knockdown.
• •Chondrocytes respond to perlecan deficiency by increasing bulk matrisome secretion.
• •Decreased stiffness may be explained by atypical glycosaminoglycan deposition.
• •Elevated COL10A1 expression and chondrocyte hypertrophy indicate early ossification.

     

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.     

Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility.     

The Sperm Proteome of the Echiuran Urechis unicinctus (Annelida,Echiura)

Sperm proteins presumably play critical roles in reproduction, but in many non‐model animals their identities are unknown. A total of 147 sperm proteins from the echiuran worm Urechis unicinctus, the first sperm proteome in the phylum Annelida, are reported. The echiuran sperm proteome can be classified into diverse functional groups: energy metabolism (31%), protein synthesis and degradation (18%), spermatogenesis and sperm motility (12%), signal pathway (11%), ion channel and transport proteins (6%), cytoskeleton (4%), immunity and stress responses (3%), and fertilization (1%). These results will facilitate studies of mechanisms of fertilization in echiurans, as well as comparative studies of reproduction and evolution across lophotrochozoans. Data are available via ProteomeXchange with identifier PXD009176.     

Soil Moisture Alters the Response of Soil Organic Carbon Mineralization to Litter Addition

Increasing rainfall and longer drought conditions lead to frequent changes in soil moisture that affect soil organic carbon (SOC) mineralization. However, how soil moisture affects response of SOC mineralization to litter addition in forest ecosystems remains unexplored. We added ¹³C-labeled litter to subtropical forest soils with three mass water contents (L, 21%; M, 33%; H, 45%). Carbon dioxide production was monitored, and the composition of soil microbial communities was determined by phospholipid fatty acid (PLFA). When no litter was added, SOC mineralization was greater in the M-treated soil. Litter addition promoted SOC mineralization, but this promotion was altered by soil moisture and litter type. Priming effects induced by P. massoniana leaf litter in the M-moistened soil were significantly (P < 0.05) higher than those in other treatments. Litter-derived C was approximately 55% incorporated into 18:1ω9c and 16:0 PLFAs, and this proportion was not significantly affected by soil moisture. Soil moisture affected the distribution of litter-¹³C in i15:0, i17:0, and cy19:0 individual PLFAs. The primed C evolution was significantly related to the ratio of Gram-positive to Gram-negative bacteria. These results suggest that changes in soil moisture could affect SOC mineralization in forest ecosystems.     

水牛精子蛋白质组双向电泳体系的建立和优化

建立和优化一种适合水牛精子蛋白质组学研究的双向电泳技术。以水牛精子为研究对象,比较两种不同配方的裂解液,以及不同上样量对其2-DE图谱质量的影响。结果显示,以7 mol/L尿素、2 mol/L硫脲、4%CHAPS、1%DTT、0.5%Cocktail of protease inhibitors为裂解液,24 cm胶条上样量200μg时,可获得较好的精子总蛋白质2-DE图谱。运用ImageMaster 2-Dplatinum分析软件检测出约500个蛋白质点,蛋白质大部分分布在等电点5-7之间,分子量范围约40-90 kD。     

Proteomic Analyses Reveal a Role of Cytoplasmic Droplets as an Energy Source during Epididymal Sperm Maturation

A small portion of cytoplasm is generally retained as the cytoplasmic droplet (CD) on the flagellum of spermatozoa after spermiation in mice. CDs are believed to play a role in osmoadaptation by allowing water entrance or exit. However, many lines of evidence suggest that CDs may have roles beyond osmoregulation. To gain more insights, we purified CDs from murine epididymal spermatozoa and conducted proteomic analyses on proteins highly enriched in CDs. Among 105 proteins identified, 71 (68%) were enzymes involved in energy metabolism. We also found that sperm mitochondria underwent a reactivation process and glycolytic enzymes were further distributed and incorporated into different regions of the flagellum during epididymal sperm maturation. Both processes appeared to require CDs. Our data suggest that the CD represents a transient organelle that serves as an energy source essential for epididymal sperm maturation.     

The Aspergillus fumigatus Secretome Alters the Proteome of Pseudomonas aeruginosa to Stimulate Bacterial Growth: Implications for Co-infection

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Highlights

• •Pseudomonas aeruginosa growth increases in Aspergillus fumigatus culture filtrates.
• •A. fumigatus culture filtrates are characterized by a range of peptidases and proteases.
• •LFQ proteomics characterizes the response of P. aeruginosa to A. fumigatus culture filtrates.
• •A. fumigatus creates an environment for P. aeruginosa to proliferate.

     

用三色荧光原位杂交(Three-Color FISH)检测小鼠精子中染色体数目异常的研究

用小鼠X、Y和8号染色体特异的DNA探针,与经DTT（dithiotreitol）和LIS（lithium-3,5-diiodosalicylicacid）解聚的小鼠附单精子进行三色荧光原位杂交（fluoresceoceinsituhybridization,FISH）,以检测精子中的染色体数目异常,并与MMⅡ染色体分析比较．结果表明精子三色FISH具有以下优点和特点：（1）方法敏感稳定,且简便快速;（2）在每一个体至少分析10000尾精子的基础上计算非整倍体单,因此结果更为准确;（3）能检测多倍体即减数分裂停止的发生率及停止的时期;（4）不仅能测定发生于试数分裂Ⅰ（MI）的染色体分离异常,还能检测发生于减数分裂Ⅱ（MII）的不分离和丢失．并对探针的选用、分析标准的建立以及三色FISH用于精于染色体分析的必要性等进行了讨论．