哺乳动物精子性别特异膜蛋白的研究进展

在精子形成过程中,存在性染色体特异基因的表达,这是X、Y精子膜蛋白差异形成的基础。尽管精子形成过程中形成的细胞间桥可能使精子细胞间共享基因表达产物,但雄性传递偏移现象和性别偏移现象的发生又证明两类精子间存在非共享蛋白,同时H-Y抗原表位的成功鉴定、精子分离实验结果以及性别特异蛋白的检测结果都肯定了精子膜蛋白差异的存在,只是这种膜蛋白的差异很小。蛋白分离技术的进步及技术间的优化集成,为精子间细微差异膜蛋白的分离提供了可能。     

人精子顶体膜蛋白的纯化及性质的研究

目的：提取和纯化与人卵透明带(HZP)作用更为密切的人精子膜蛋白,为改进精子包被抗原的制备方法和筛选避孕疫苗抗原提供科学依据。方法：将人精子膜蛋白混合液进行SDS-PAGE电泳,然后全胶电洗脱分剐收集蛋白溶液30管,以阳性管内的HZP特异精子蛋白溶液包被抗原进行AsAb检测。结果：与HZP结合的人精子膜蛋白有多种成分;全胶电洗脱后以第14管内成分之特异最强,其分子量范围为48～53KD。结论：以电洗脱转移法纯化筛选的人精子膜蛋白是特异的,与HZP具极强相关性,可以用该精子膜蛋白包被抗原进行AsAb研究。     

一种与受精有关的人精子膜蛋白（BS—17）的分离纯化

本文盐分级分离,Ｍｏｎｏ Ｑ ＦＰＬＣ及ＳＤＳ－聚烯酰胺胶电泳（ＳＤＳ－ＰＡＧＥ）等方法从人精子ＣＨＡＰＳ抽提液中分离纯化出一种与不育病血清中抗精子抗体发生特异反应的ＢＳ－１７人精子膜蛋白。该蛋白为一糖蛋白,分子量为１７．５５±２．１５ｋＤ,等电点为５．６５,中性己糖含是为１６．６７％。在人精子上主要分布于顶体区域,不同于已有报导的人精子膜蛋白。在体外实验中抗ＢＳ－１７多克隆血清可以显著影响人精子     

玉米精细胞质膜特异蛋白的纯化

在获得6个品种的玉米(Zea mays L.)花粉精细胞后,采用N-hydroxysuccinimido-biotin(NHS-biotin)标记其外膜蛋白,并通过SDS-PAGE和Western blotting对比了其中主要标记蛋白,发现其主要蛋白带差异并不显著,主要标记蛋白分子量均集中于91、60、43、30和17kD。采用免疫亲和层析技术进一步纯化已获得的混杂少量其它细胞器成分的精细胞质膜制剂,即利用制备的体细胞主要细胞器:线粒体、内质网、高尔基体及质膜的膜蛋白,分别免疫豚鼠,从其抗血清中纯化获得IgG,并进一步制成各种膜蛋白的免疫亲和吸附制剂。利用此技术进一步纯化经NHS-biotin标记的精细胞质膜蛋白,获得精细胞质膜特异的蛋白质,其中最为显著的蛋白质分子量约为65、22kD。     

一种精子表面GPI锚定蛋白初步鉴定方法的建立

精子表面糖基磷脂酰肌醇（GPI）锚定蛋白(GPI-Aps)在精子不同阶段都发挥着关键作用.本研究介绍了嗜水气单胞菌毒素（aerolysin）作为生物探针来鉴定精子表面的GPI锚定蛋白.实验依次进行了嗜水气单胞菌培养、毒素提取纯化、多克隆抗体制备以及三明治蛋白印迹验证实验.凝胶蛋白分离实验显示,硫酸铵沉淀加Sephadex G-100层析柱纯化得到了分子质量为45 kD高纯度细菌毒素|蛋白印迹实验验证了制备的多克隆抗体的特异性|三明治蛋白印迹结果显示,嗜水气单胞菌毒素能识别出12条精子脂筏提取蛋白条带,其分子质量主要分布在15~130 kD之间|Alex488标记的免疫荧光实验显示,嗜水气单胞菌毒素识别的GPI锚定蛋白主要分布在精子头部和尾部.研究结果提示,嗜水气单胞菌毒素能够有效地识别精子表面的GPI锚定蛋白.     

一种与受精有关的人精子膜蛋白（BS－17）的分离纯化

本文用盐分级分离,ＭｏｎｏＱＦＰＬＣ及ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）等方法从人精子ＣＨＡＰＳ抽提液中分离纯化出一种与不育病人血清中抗精子抗体发生特异反应的ＢＳ－１７人精子膜蛋白。该蛋白为一糖蛋白,分子量为１７．５５±２．１５ｋＤ,等电点为５．６５,中性己糖含量为１６．６７％。在人精子上主要分布于顶体区域,不同于已有报导的人精子膜蛋白。在体外实验中抗ＢＳ－１７多克隆血清可以显著影响人精子的获能（ｐ＜０．０２５）和对去透明带仓鼠卵的穿透（ｐ＜０．００５）,但不影响人精子运动性及与去透明酯酸带仓鼠卵的结合。小鼠体内被动免疫实验结果证明抗ＢＳ－１７多抗血清具有明显地抑制受精的功能（ｐ＜０．００１）。     

草鱼血清IgM蛋白的纯化及抗血清的制备

采用盐析法、重组蛋白A(HiTrapr Protein A Sepharose)亲和层析法分离纯化草鱼血清中的IgM,并通过SDS-PAGE及Western-blot技术对纯化蛋白的部分特性进行分析比较并制备兔抗IgM抗血清。结果表明:33%硫酸铵溶液可以沉淀血清中大部分蛋白,但电泳条带仍较多,其中含有78kD和28kD的条带,因此仅可作为免疫球蛋白粗提的方法;而rProtein A亲和层析法所提蛋白则仅有上述重链(78kD)和轻链(28kD)。Western-blot显示,鼠抗人Ig抗体可与78kD及28kD条带发生发应。rProtein A亲和法提纯蛋白的纯度较高,但含量较低,条带较淡,仅可作为实验室小量提纯草鱼IgM的有效方法。将提纯的蛋白免疫实验兔后可制得效价高达1:25600的兔抗鱼IgM血清,并测得血清蛋白总量和IgM含量分别为25.87mg和4.5mg,IgM占血清蛋白总量的17.39%。本实验所采用的蛋白A亲和层析法提取草鱼血清IgM可以方便、快捷地获得高纯度的产物,适合在实验室中纯化鱼类IgM。同时本研究所制备的兔抗草鱼IgM血清也为今后的相关研究工作打下基础。     

双歧杆菌肽聚糖结构及分子量的分析

从双歧杆菌细胞壁中分离纯化肽聚糖,研究其化学成分、结构和分子量分析。经氨基酸、多糖和蛋白质含量分析和肽聚糖的溶解实验鉴定,所提取物质的主要成分为肽聚糖;经核磁共振和红外光谱分析,所提取肽聚糖的结构与已知的肽聚糖的结构相吻合,其糖链结构为由D型吡喃糖N-乙酰葡糖胺和N-乙酰胞壁酸以β-1,4-糖苷键连接而组成;经SDS-PAGE和多种染色方法结合,分析经溶菌酶水解后的肽聚糖,其分子量的分布呈弥散状,范围在97.6kD到14.4kD之间。     

黑木耳漆酶纯化的研究

目的:研究黑木耳(Auricularia auricula)“黑29”的漆酶纯化,为进一步酶学性质的开展、酶应用和酶基因克隆提供理论基础。方法:采用硫酸铵分级沉淀和柱层析技术分离蛋白,通过PAGE和SDS-PAGE电泳检测蛋白。结果:SDS-PAGE电泳检测发现粗酶液含三种漆酶,分子量大小分别为LacA(60kD)、LacB(34kD)、LacC(19kD);纯化后获得纯化的单一漆酶LacA、LacC组分。结论:得漆酶两个单一组分,为进一步漆酶研究奠定基础。     

棉铃虫中肠微粒体P450的分离纯化

为深入研究棉铃虫Helicoverpa armigera细胞色素P450的结构与功能,需要分离不同型的P450蛋白。作者建立了适用于棉铃虫中肠微粒体P450的纯化方法,包括聚乙二醇8000（PEG8000）沉淀、高效疏水作用色谱（HPHIC）和高效离子交换色谱（HPIEC）等连续分离步。SDS-PAGE（银染）显示,棉铃虫中肠微粒体经以上步骤分离纯化后,在含P450的馏分中检测出分子量分别为58 kD、47 kD、56 kD和45 kD的4条蛋白带。 P450的回收率为14.3%,比含量提高了39倍。     

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis

The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1,000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation. © 1996 Wiley-Liss, Inc.     

Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.

Nucleoplasmin is necessary and sufficient for the initial stage of Xenopus sperm decondensation in egg extracts. In this article we show that sperm decondensation is accompanied by loss of two sperm-specific basic proteins (X and Y) and gain of histones H2A and H2B, resulting in nucleosome formation. Purified nucleoplasmin alone removes X and Y and assembles purified H2A and H2B on decondensing sperm chromatin, forming nucleosome cores. Immunodepletion of nucleoplasmin from extract prevents removal of X and Y and addition of H2A and H2B, while adding back nucleoplasmin restores decondensation and X and Y removal. Thus, nucleoplasmin acts as both an assembly and a disassembly factor for remodeling sperm chromatin at fertilization.     

Purification of bicarbonate-sensitive sperm adenylylcyclase by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-affinity chromatography.

Anion transport inhibitors, such as SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) and heparin, inhibit reversibly the bicarbonate-sensitive adenylylcyclase of porcine sperm plasma membrane. In the light of this, SITS- and heparin-affinity chromatographies were applied in order to purify sperm adenylylcyclase. SITS-Affi-Gel 102 binds proteins extracted from the porcine cauda epididymal sperm plasma membrane by Lubrol-PX, more selectively than heparin-agarose. However, recovery of adenylylcyclase activity is higher when heparin-agarose is used. The hormone-sensitive liver adenylylcyclase, which is less sensitive to bicarbonate than sperm enzyme, has less affinity for these affinity resins than sperm enzyme. Adenylylcyclase can be purified to apparent homogeneity on two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis) from the Lubrol-PX extract of the purified sperm plasma membrane by using SITS-affinity chromatography at the first step of the purification followed by preparative isoelectric focusing and gel filtration. The molecular weight and pI of the purified enzyme are 46,300 and 6.9, respectively. The purified enzyme activity is highly dependent on Mn2+. Bicarbonate activates even the purified enzyme both by decreasing Km and by increasing Vmax.     

兰州百合精细胞特异蛋白的研究

通过低渗冲击及Percoll密度梯度离心的方法,成功地分离并纯化了兰州百合(Lilium davidiiDuch.)生活的生殖细胞及精细胞。从精细胞、生殖细胞及叶片中提取了全蛋白,并通过双向电泳技术对它们进行了比较。在双向电泳图谱上精细胞比生殖细胞显示更多的蛋白斑点,特别是在碱性端。通过混合酶解及离心,分离了生活的叶肉原生质体。用生物素的琥珀酰胺酯衍生物(NHS-biotin)对精细胞、生殖细胞及完整的叶肉原生质体质膜蛋白进行标记,然后进行Western blot分析,用辣根过氧化物酶酶标链霉抗生物素蛋白及其底物4-氯-1-萘酚反应显色,比较了3种质膜蛋白。发现分子量为46kD及50kD的两种蛋白是精细胞质膜特异的。在双向电泳图谱上也可找到与这两种蛋白相对应的斑点,它们很可能与受精过程中精卵的识别有关。     

Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.     

Comparison of Membrane Surface Proteins of Sperm Cells and Somatic Protoplasts of Zea mays

Viable sperm cells and somatic protoplasts (leaf, callus) of Zea mays were successfully isolated and purified. The plasma membrane surface proteins of intact somatic protoplasts and sperm cells were compared after probing with N-hydroxysuccinimido-biotin (NHS-bi-otin). Horseradish peroxidase-labelled avidin (HRP-avidin) was used to detect membrane proteins after separation by SDS-PAGE and Western blot. Four protein bands characteristic of the surface membrane of sperm cells were identified varying from 48 to 78 kD, five bands of leaf protoplasts in the range of 45~78 kD, and two bands of callus protoplasts, 67 and 80 kD were detected. One protein of 48 kD was specific to the surface membrane of sperm cells and might be related to the specific roles of sperm cell physiology.     

Biochemical characterization of two ram cauda epididymal maturation-dependent sperm glycoproteins

Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.     

Maturation-dependent modification of the protein phosphorylation profile of isolated goat sperm plasma membrane.

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca(2+)-calmodulin, phosphatidylserine-diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.     

Identification of calcium conducting channels in isolated boar sperm plasma membranes

Ion channel recordings were obtained from liposomes containing purified boar sperm plasma membrane proteins using a tip-dip method. Liposomes prepared in HEPES-TRIS and clamped by electrodes containing Ba-HEPES displayed channel activity that was partially inhibited by verapamil or nitrendipine and completely inhibited by La3+. Reversal of current at pipette negative voltages was observed only when Ba2+ ions were also present in the bath solution. These data indicate that channels capable of carrying calcium currents are prominent components of the plasma membrane of mammalian sperm.     

Identification of heterotrimeric G proteins in human sperm tail membranes

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α-subunits, and A 86, which detects all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein α-subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa β₁-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive α-subunits. However, membrane preparations of nonpurified human spermatozoa contained α₂ subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α-subunits; the putative β-subunit was identified as a β₂-subunit. © 1995 Wiley-Liss, Inc.