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.

     

Purification and Characterization of Kininogens from Sheep Plasma

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep ( Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. K_i values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. K_i values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.     

Purification and Partial Characterization of Hypocalcemic Factor from Human Saliva

This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chromatography. A second gel filtration on Sephadex G-150 separates two minor salivary protein contaminants (IgA and IgG immunoglobulin) in the excluded fraction from the smaller, hypocalcemically active polypeptide.

No hypocalcemia activity could be detected or isolated in a preliminary investigation on the saliva of a dysgammaglobuli-nemic (IgA deficient) patient.

The hypocalcemia induced does not differ significantly from that observed after administration of calcitonin to mice in that: 2) minimum values are reached in 1.5–2 hours and return to normal in 5–6 hours, b) magnitude of hypocalcemia response is dose dependent. The salivary hypocalcemia factor isolated in this study has the properties of a protein, in that its activity is destroyed by the proteolytic enzyme trypsin, it yields amino acids upon acid hydrolysis and it behaves on electrophoresis, gel filtration and ion exchange chromatography as a typical protein.     

Purification and Amino-terminal Sequencing of a 68 kD Glycopolypeptide of the Plasma Membrane from Maize Sperm Cells

Based on author's previous work on detection and immunolocalization of glycoproteins of the plasma membrane of maize ( Zea mays L. ) sperm cells, a 68 kD peripheral specific glycopolypeptide of the plasma membrane from maize sperm cells was purified by IEF-SDS two-dimensional electrophoresis. It presents specif- ically positive reaction in Con A-HRP (concanavalin A-horseradish peroxidase) staining, and its pi value is 5.5. The search in protein sequence database reveals that the amino-terminal sequence of this glycopolypeptide is identical with that of Con A. But its difference from Con A in molecular weight and pi value indicates that it could be related to a Con A receptor on the plasma membranes of maize sperm cells instead of being Con A itself. It is fascinating to study further the function of the above glycopolypeptide in gametic recognition, adhesion and fusion of the double fertilization in maize.     

Characterization and Partial Purification of a Novel Neuronotrophic Factor from Bovine Seminal Vesicle

Extracts from bovine seminal vesicles have been shown to contain high concentrations of nerve growth factor (NGF)-like biological activity and of the NGF protein with properties corresponding to that of NGF from other sources. We now demonstrate that a second neuronotrophic protein, termed seminal vesicle-derived neuronotrophic factor (SVNF), is present in seminal vesicle extracts (SVEs), which could not be distinguished from NGF on the basis of biological activity. SVNF has neuronotrophic activity on NGF target cells like embryonic chicken-sensory and sympathetic neurons, sympathetic neurons, and chromaffin cells from neonatal rats, but it is inactive on embryonic chicken ciliary or neonatal rat nodose ganglion neurons. It also stimulates fiber outgrowth from rat pheochromocytoma (PC 12) cells. In gel filtration chromatography on Biogel A 1.5 m, the activity is eluted with an apparent molecular weight of 40 kilodaltons, and by preparative isoelectric focusing, the isoelectric point was determined to be in the neutral range (6.8-7.8). The biological activity of SVNF, in contrast to that of NGF, is partially retained after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can be electrophoretically eluted with an apparent molecular weight of 16-20 kilodaltons. Electrophoretically purified SVNF is not inhibited by antisera to mouse NGF, but its activity is increased greater than 10-fold in the presence of very low concentrations of NGF. For partially purified SVNF, a specific activity of 2.9-5.8 X 10(5) biological units/mg of protein was determined in the presence of subthreshold NGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

尖吻蝮蛇毒内一种新的抗血小板凝集蛋白agkisacutacin的纯化及其性质

从皖南尖吻蝮蛇毒中经阴离子交换层析和凝胶过滤层析分离纯化得到抗血小板凝集蛋白agkisacutacin ,纯化的agkisacutacin由分子量为 1 4kD和 1 5kD的 2条肽链通过二硫键连接 ,能有效抑制ristocetin诱导的血小板凝集 (IC5 0 为 1 8.5mg/L) ,能轻微抑制凝血酶诱导的血小板聚集 (IC5 0 为 1 .2 2 g/L) ,但对ADP、胶原诱导的血小板聚集无影响。agkisacutacin不具有纤溶活性、抗凝活性、磷脂酶A2 活性和出血活性 ,是一种潜在的安全、有效的抗血小板性栓塞的药物     

猪精子凝集素的纯化,性质及其作用

用胎球蛋白－Ｓｅｐｈａｒｏｓｅ亲和层析和凝胶过滤层析从精子和精浆中分离纯化了猪精子凝集素（简称ＢＳＬ）。ＢＳＬ的血凝活性只被若干糖蛋白和聚糖所抑制。ＢＳＬ的分子量为５６ｋｄ,由分子量分别为１３．６ｋｄ（β）和１６．０ｋｄ（α）的两个不同的亚基以α１β３所组成。ＢＳＬ为糖蛋白,含中性糖３．２％,不含唾液酸。用ＥＬＩＳＡ法测定猪精子中ＢＳＬ的含量及分布,表明７０％嵌入在精子膜中,２５％结合在精子表面,     

人前列腺生长因子的分离纯化及生物活性鉴定

从人良性增生前列腺组织中经硫酸铵沉淀和肝素－琼脂糖凝胶层析纯化出人前列腺生长因子（ｈＰＧＦ）,纯化倍数约１０００倍,ＳＤＳ－ＰＡＧＥ和等电聚焦电泳示分子量约为１７ｋＤ、等电点同标准ｂＦＧＦ,利用分离培养的人前列腺间质成纤维细胞进行活性鉴定,发现以１．３～１．７ｍｏｌ／ＬＮａＣｌ洗脱部分为ｈＰＧＦ,活性最高,对间质成纤维细胞有显著刺激增殖作用。     

巨噬细胞源成纤维细胞生长因子的分离纯化

为探讨二氧化硅在大鼠肺内引发的一系列纤维增生反应,试图寻找一种关键的细胞增生因子,采用高压液相色谱技术,包括凝胶过滤柱层析、离子交换柱层析以及反相Ｃ４高压液相色谱柱层析,获得一种新的巨噬细胞源成纤维细胞生长因子（ａｌｖｅｏｌａｒｍａｃｒｏｐｈａｇｅ－ｄｅｒｉｖｅｄｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃ－ｔｏｒ,ＡＭＤＧＦ）,ＳＤＳ－ＰＡＧＥ结果表明它已达到均一的纯度,其分子量为５８０００,ｐＩ为４．７．该因子的分子量与ＭΦ在体外接受石英粉尘刺激分泌的因子的分子量明显不同．Ｎ端序列测定结果显示它与已知蛋白序列的同源性小于５０％,其刺激成纤维细胞增殖的最适浓度范围为２．０～６．０μｇ／ｍｌ,推测ＡＭＤＧＦ是矽肺纤维化病变发生、发展过程中调节成纤维细胞增殖的重要因子之一     

Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.     

Purification and Characterization of a Cytostatic Factor with Anti-Viral Activity from the Bitter Melon

Abstract

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activi-ties ^1,2, and is a competitive inhibitor of guanylate cyclase activity³. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses¹.

In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity.

The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This pre-paration was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons.

The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse).

As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system⁴. DNA synthesis was slightly stimulated.

The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h.

The cytostatic component had no effect on cellular cyclic GMP metabolism.     

Optimum Calcium Concentration: A Crucial Factor in Regulating Sperm Motility In Vitro

Sperm motility can be maintained in vitro by incubation in a defined medium under specific conditions. In most studies, the exact role of various constituents of epididymal fluid, including calcium, has remained obscure. Most of the culture media have included millimolar concentrations of calcium, but previous reports have indicated that millimolar calcium inhibits sperm motility. In this present study, we sought the optimum concentration of extracellular calcium required for optimum sperm motility. This study showed that extracellular calcium has a concentration-dependent biphasic role in motility regulation. It promoted motility and velocity at lower (10 µM) concentration whereas notably inhibited it at higher concentrations. When external membrane-bound calcium was removed by ethylene glycol tetraacetic acid, motility decreased considerably. To confirm the motility-inhibiting role of calcium above 10 µM, a sperm motility-stimulating protein (MSP) recently reported from our laboratory was used which at 0.9 μM induces motility in 60–70 % cells. Calcium at 10 µM had no appreciable effect on the motility-promoting activity of the MSP but depressed the activity above 10 µM. Thus, our present results emphasize the biphasic role of extracellular calcium and the importance of its optimum concentration in different buffers and media used for sperm motility initiation.     

A Neurotrophic Factor Derived from Goldfish Brain: Characterization and Purification

Previous studies carried out in our laboratory have demonstrated that goldfish brain contains substances that promote neurite extension from regenerating retinae in culture. Fractionation of the brain extract by molecular sieving chromatography revealed the presence of several molecular species, including two peaks that have neurotrophic activity, representing low-molecular-weight substances. One peak was eluted (P-a) with an apparent molecular weight of about 13 kDa and was designated substratum neurite extension factor (SNEF) because it retained its neurotrophic activity when adsorbed onto the substratum. This recovered Sephadex fraction (P-a) when applied in vivo intraocularly caused an earlier capacity of the corresponding retinae to sprout in vitro. Thus, at 3 and 5 days after injury the neuritic growth indices from the factor-treated retinae were of 0.9 +/- 0.2 and 2.8 +/- 0.5, respectively, as compared with indices of 0.3 +/- 0.1 and 0.9 +/- 0.2, respectively, in retinae of injured but nontreated nerves. The factor was further purified by two steps of HPLC (ion exchange followed by reversed phase). The results showed that it is an acidic glycoprotein with an apparent molecular weight of 10 kDa.     

The Effect of Low-Level Laser Irradiation on Sperm Motility,and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm

Background and Objective

Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm.

Study Design/Materials and Methods

We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS - Integrated Semen Analysis System.

Results

Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05).

Conclusion

We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was more effective than a dose of 6 joules in preserving sperm motility.     

人血浆蛋白C的分离纯化与鉴定

冰冻人血浆37℃融化后,经钡盐吸附沉淀、硫酸铵分步盐析、DEAE-Sephadex A-50柱层析、制备性PAGE和肝素亲和层析分离,得到蛋白C。经鉴定,所得蛋白C分子量约为62kD,pI为4.69,PAGE分析高度均一,KPTT法证实其延长凝血时间,与有关文献报道相符。     

牛脑成纤维细胞生长因子的分离纯化与鉴定

新鲜牛脑组织匀浆液经两步硫酸铵沉淀、CM-Sephadex C50 离子交换层析以及肝素-Sepharose 亲和层析,可得到纯化的酸性和碱性成纤维细胞生长因子(aFGF 和bFGF),分子量分别为13.2kD 和15.2—15.8kD.两种因子均可有效促进3T3细胞的 DNA 合成,ED₅₀分别为15.8ng/ml 和 0.32ng/ml.进一步对 aFGF 的等电点及氨基酸组成做了分析.     

蕲蛇蛇毒抗凝血因子的分离纯化与特性

目的 寻找蕲蛇蛇毒中的抗凝血因子。方法 利用硫酸铵沉降、阴离子交换层析、阳离子交换层析及高效液相色谱层析,从蕲蛇蛇毒中分离纯化到一个抗凝血因子。结果 纯化的这一组份在PAGE、SDS—PAGE上均呈单一区带,分子量约为25．4kD,由两条分子量分别为15．0kD和16．0kD的肽链通过二硫键连接在一起。这一组份在体外显著地延长血浆复钙时间和凝血酶原时间,但不延长牛凝血酶时间,也不具有磷脂酶A2活性、纤溶活性和出血活性。结论 蕲蛇蛇毒中舍右一种新的抗凝血因子。     

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.     

Purification of the Specific Plasma Membrane Proteins from the Surface of Sperm Cells of Zea mays

Pollen samples of 6 varieties of Zea mays L. were used to isolate the viable sperm cells. After being probed with N-hydroxysuccinimido-biotin (NHS-biotin), the sperm cell plasma membrane proteins were compared with each other using the method of Western blotting. Results showed that there was no significant difference among varieties. The molecular weights of probed plasma membrane proteins were concentrated on 91,60,43,30 and 17 kD. Immunochemical method was adopted for further purification of sperm plasma membrane protein preparation which was some- what contaminated with cell organelles. After the cell organelles were isolated from etiolated seedlings of Zea mays by sucrose density gradient super centrifugation, the crude membrane proteins of organelles, endoplasm reticulum, mitochondria, Golgi body and plasmolemma were respectively used as antigen to immunize Guinea pig. The antibody was obtained from respective antiserum, then further used to produce immuno-affinity absorbent. After the solution of membrane proteins of sperm cells passed through the column, some proteins probed whth NHS-biotin were identified. Two major proteins probed with NHS-biotin were considered to be sperm cell specific. The size of these proteins in SDS-PAGE was about 65 kD, 22 kD, respectively.     

Construction,Purification, and Characterization of a Homodimeric Granulocyte Colony-Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) has found widespread clinical application, and modified forms with improved biopharmaceutical properties have been marketed as well. PEGylation, the covalent modification of G-CSF with polyethylene glycol (PEG), has a beneficial effect on drug properties, but there are concerns connected to the immunogenicity of PEGylated compounds and bioaccumulation of the synthetic polymer. To overcome challenges connected with chemical modifications, we developed fusion proteins composed of two G-CSF molecules connected via different peptide linkers. Three different homodimeric G-CSF proteins were purified, and their in vitro and in vivo activities were determined. A G-CSF dimer, GCSF-Lα, was constructed using an alpha-helix-forming peptide linker, and it demonstrated an extended half-life in serum with a stronger neutrophil response as compared to the monomeric G-CSF protein. The GCSF-Lα protein, therefore, might be selected for further studies as a potential drug candidate.