哺乳动物精浆生化指标对精液品质的影响

为了研究哺乳动物精浆生化指标对精液品质的影响,试验通过分析精浆糖类、蛋白质、微量元素对精液品质的影响,阐明了精浆成分对动物精液品质的重要作用。结果表明:精浆糖类是精子活动的能量来源;精浆中的蛋白质对顶体反应、精子获能、精子贮存、精卵识别和受精等功能具有显著影响;微量元素可直接参与多种酶促反应和蛋白质的结构组成。     

17℃保存对猪精子亚组分蛋白酪氨酸磷酸化的影响及中药保护作用

实验旨在分析17℃保存对获能精子蛋白酪氨酸磷酸化的影响,通过精子蛋白亚组分分离及酪氨酸磷酸化鉴定,研究精浆以及中药水提液对保存精子获能相关蛋白酪氨酸磷酸化的影响。选择获能指标-酪氨酸磷酸化分析新鲜猪精子和17℃保存猪精子,提取精子全蛋白、膜蛋白、核蛋白以及骨架蛋白,利用WB分离及鉴定酪氨酸磷酸化差异。结果显示:17℃保存条件下,获能精子在40～45、46～50 ku分子量膜蛋白酪氨酸磷酸化程度减弱;未获能精子在30～35 ku分子量胞浆蛋白磷酸化程度增强,出现似"获能"现象;精浆在保存中起到提供营养的作用但易导致细菌滋生;中药水提液对精子保存过程中的保护效果体现在精子获能培养后,鞭毛中段、主段酪氨酸磷酸化和新鲜精子无明显差异。     

哺乳动物成熟精子的转录及翻译活性研究进展

精子形成过程中存在经典的"转录-翻译解偶联"现象.精子变形过程中将大量RNA贮存在核糖体颗粒蛋白RNPs和多聚核糖体中,以便在特定发育阶段被激活翻译.近年来的研究表明,获能反应以及冷冻、性别筛选、外源添加剂孵育等体外操作均会改变成熟精子的蛋白质组,线粒体作为精子获能过程中部分蛋白翻译的场所,具有重要作用.本文围绕成熟精...     

精浆外泌体的转运机制及生物学功能研究进展

精浆外泌体是由雄性性腺及副性腺上皮细胞分泌至雄性生殖道中,直径为30～100 nm的膜性小囊泡,其构成精子发育的主要环境,是精子成熟过程中不可或缺的一部分.精浆外泌体中富含蛋白质、脂质、核酸等物质,这些物质通过独特的转运机制被运送至精子表面以促进精子形态与功能的完善,精浆外泌体在提高精子活力、抑制精子提前获能、抗精子氧...     

羊精浆蛋白与精子功能相关性研究进展

射精过程导致精子与精浆相互汇合,精浆为精子的体内受精和体外保存提供保护作用。精浆中的蛋白可以作为预测精子功能和生育能力的标志物。蛋白质组学技术的不断发展为更深入地研究精浆蛋白对精子生理功能的影响及其相关作用机理提供了新的技术支撑和研究方法。文章综述了利用蛋白组学对羊精浆蛋白的部分研究结果,以期明确羊精浆蛋白与精子功能的相关性,为今后利用精浆蛋白提高精子品质及保存效果奠定一定的理论基础。     

水牛精子活力相关的精浆外泌体差异蛋白质组学研究

为探索水牛高低活力精子的差异表达蛋白质（Differentially Expressed Proteins,DEPs）变化，并揭示水牛精子活力相关蛋白质的分子调控机制，本研究通过液相色谱-串联质谱（LC-MS/MS）技术，对成年摩拉水牛精浆、精子、精浆外泌体蛋白质种类及表达量进行比较分析。按照精子活力分为高活力组（0.67±0.02）和低活力组（0.34±0.04），分别提取其精浆外泌体蛋白质进行差异表达分析，鉴定到DEPs共160个。通过GO分析和KEGG通路分析发现，DEPs显著富集到精子部位的顶体素结合蛋白（ACRBP）、精子发育相关的生物调节过程以及“FcγR介导的吞噬作用”、“PPAR信号通路”等多个信号通路。通过构建精浆外泌体DEPs互作网络进一步分析发现，ACRBP、IZUMO1、SPACA1、ADAM2等蛋白质在精子发育调控通路发挥重要作用。     

哺乳动物获能精子蛋白酪氨酸磷酸化研究进展

哺乳动物成熟精子是一种高度分化的生殖细胞,"获能"后具有受精能力,其中酪氨酸磷酸化过程是获能过程中非常重要的环节,参与调控获能相关的顶体反应和超激活运动。精子获能分子机理研究有助于解决临床男性不育以及男性避孕问题,同时为牲畜体外人工授精技术应用提供理论支持。目前哺乳动物获能精子蛋白酪氨酸磷酸化的分子机制研究主要集中在信号通路、蛋白质种类鉴定、影响因素等方面。     

获能前后绵羊精子的差异蛋白质组学分析

为了探索获能前后绵羊精子蛋白质的动态变化，通过串联质谱标签结合液质联用分析技术，对获能前后绵羊精子的总蛋白进行定量分析。结果显示：获能前后绵羊精子的差异表达蛋白共有203个，获能后表达上调蛋白质27个，表达下调蛋白质176个。差异表达参与解剖结构发育、生殖等生物学进程；从细胞组分看，差异表达蛋白定位在细胞溶质、质膜中；从分子功能看，主要参与氧化还原等过程。差异表达蛋白也参与蛋白酶体、Hedgehog和cAMP等信号通路。通过差异蛋白表达结合文献分析，筛选出乳铁蛋白。Western blot结果表明，乳铁蛋白在绵羊精子中存在，且在获能后表达量极显著降低(P<0.000 1);RT-qPCR结果表明，乳铁蛋白在获能前后绵羊精子中均存在，且差异极显著(P<0.001),说明乳铁蛋白对绵羊精子获能有一定的影响。     

公猪精清蛋白质与受精过程的关系

精清的蛋白质由精囊腺、前列腺、附睾腺、尿道球腺等附性腺分泌，对受精作用至关重要，并且与雄性个体的繁殖力具有相关性。本文综述了与公猪受精相关的精清蛋白，阐述了精清蛋白质与受精过程中精子活力、精子获能、顶体反应等的关系以及在养猪生产中作为受精能力生物标记的应用前景。     

猪PSP-Ⅰ/PSP-Ⅱ的生物学特性研究

精浆是精子悬浊在精液中的液体,是附睾和雄性性腺分泌的相当复杂的混合物。精浆的蛋白质组成因不同的家畜而异,在不同的家畜上的报道表明,精浆中包含的蛋白质因子不仅影响精子的受精能力,还在影响雌性动物的繁殖生理学方面发挥重要的作用〔1~2〕。受精起始于精子表面的凝集素与卵母细胞透明带寡聚糖的相互作用。公猪的精浆蛋白是一个糖蛋白家族,它们的分子量在12~16kDa之间。AQN-1、PSP-I、PSP-Ⅱ、AQN-3和AWN(1和2)是精囊腺上皮细胞主要分泌物,他们占精液中总可溶性蛋白的80%〔3,4〕。另外,AWN-1由睾丸网和直精小管上皮细胞合成。…     

Characteristics of selected seminal plasma proteins and their application in the improvement of the reproductive processes in mammals

Understanding the biochemical processes associated with ovum fertilization and knowledge about the structure and function of individual substances participating in these processes is crucial for the development of biotechnological methods to improve reproduction of animals and humans. Among many components of seminal plasma, proteins and peptides play a specific role in regulation of the fertilization process, particularly through their ability to bind various types of ligands such as polysaccharides, lipids and ions. Heparin-binding proteins regulate capacitation and acrosome reaction processes. Affinity of plasma proteins to mannans of the fallopian tube epithelium facilitates formation of spermatozoa reservoirs in the female reproductive tract. Ability to bind phosphorylcholine is one of the conditions for the coating of the seminal plasma proteins on the sperm membrane and also determines the formation of oligomeric forms of certain proteins. Zinc binding by seminal plasma proteins regulates sperm chromatin condensation state. It also affects motility of these cells and acrosome reaction. The interspecies analysis indicates significant structural and functional similarities, especially for the proteins with low molecular weight. Fertility associated proteins (FAPs) have been determined in the bull, stallion, boar, ram and dog. The contents of these proteins correlate with the indicators of the fertilizing abilities of sperm. In humans, several seminal plasma proteins were found which serve as diagnostic markers of spermatogenesis, seminiferous epithelium state, and azoospermia. To determine the semen ability for preservation, measurement of some seminal plasma protein content may also be used. Addition of specific plasma proteins to a spermatozoa solution undergoing the process of preservation may be used to retain the features of the cells responsible for efficient fertilization.     

Seminal plasma components in camelids and comparisons with other species

Camelid semen is characterized by a highly viscous, low-volume ejaculate with a low concentration of spermatozoa that exhibit low progressive motility. The viscous seminal plasma is currently the major impediment to the development of assisted reproductive technologies (ARTs) in camelids. To advance ARTs such as sperm cryopreservation and artificial insemination in camelids, it is necessary to identify the cause of the viscosity and gain an understanding of the role of seminal plasma components on sperm function and fertility. Numerous compounds and proteins have been identified as mediators of sperm function and predictors of fertility in other livestock species, and understanding the importance of specific proteins has progressed the success of ARTs in these species. Current knowledge on the components of camelid seminal plasma is outlined, together with the implications of these components for the development of ARTs in camelids. The cause of semen viscosity, as well as proteins that are present in camelid seminal plasma, is described for the first time. Seminal plasma components are compared with those of other species to hypothesize their role in sperm function and fertility.     

Label‐free proteome of water buffalo (Bubalus bubalis) seminal plasma

The study aimed to describe the Bubalus bubalis seminal plasma proteome using a label‐free shotgun UDMS^E approach. A total of 859 nonredundant proteins were identified across five biological replicates with stringent identification. Proteins specifically related to sperm maturation and protection, capacitation, fertilization and metabolic activity were detected in the buffalo seminal fluid. In conclusion, we provide a comprehensive proteomic profile of buffalo seminal plasma, which establishes a foundation for further studies designed to understand regulation of sperm function and discovery of novel biomarkers for fertility. MS data are available in the ProteomeXchange with identifier PXD003728.     

Cytochrome c upregulation during capacitation and spontaneous acrosome reaction determines the fate of pig sperm cells: linking proteome analysis

To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the well-established markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose- and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization.     

In vitro assessment of semen for the prediction of fertility

The prediction of fertility is a primary goal in the field of reproductive medicine. The aim of the present paper is to describe the value of conventional and modern sperm analysis systems considering the process of fertilization. The classical assessment of motility and morphology enables the rough estimation of semen quality in order to select ejaculates for the use in artificial insemination. Recent methods for sperm diagnosis, such as fluorescent marking for the detection of sperm plasma membrane integrity, the hypoosmotic swelling test, and computer assisted semen analysis allow for the evaluation of a large number of spermatozoa and the assessment of sperm dynamics under in vitro-fertilization conditions. The oocyte penetration test investigates the ability of spermatozoa for capacitation, hyperactivation and acrosome reaction in vitro. The amount of specific seminal plasma proteins is related to fertility and thereby provides an additional semen evaluation method. For the use of a given semen test the specific in vitro condition has to be considered. In addition, the evaluated criteria relevant for the process of fertilization need to be defined. The combination of selected semen tests gives a higher accuracy for the prediction of fertilizing capacity compared with a single test.     

New strategies of boar sperm cryopreservation: Development of novel freezing and thawing methods with a focus on the roles of seminal plasma

Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.     

Antisperm responses in male dogs with chronic Brucella canis infections

Male Beagles infected with Brucella canis for greater than or equal to 3 months developed serum antibodies that agglutinated normal canine spermatozoa. Titers were highest in dogs that had been infected for 4 to 6 months. Lower spermagglutinin titers were detected in sera collected 10 months after inoculation. Antibodies were also observed in seminal plasma of chronically infected dogs. Seminal plasma from infected, but not from clinically normal dogs, caused head-to-head agglutination of normal sperm. In contrast to macroagglutination of sperm by serum antibodies, agglutination by seminal plasma antibodies was detected only by microscopic examination. Seminal plasma agglutinins were not inactivated by heat (56 C, 1 hour) or by reduction with 2-mercaptoethanol. When seminal plasma and sperm were mixed with 2 hemolytic units of guinea pig complement, spermatozoa were not inactivated. Spermagglutinin activity was present in the first 2 spectral absorption peaks of Sephadex G-200 fractionated seminal plasma. Fractions that had the highest spermagglutinin titers contained mostly immunoglobulin A. Seminal plasma from infected dogs also contained cytophilic factors for normal splenic macrophages that caused sperm adherence to macrophages. Dogs with a bacteremia lasting greater than 4 months had cutaneous delayed-type hypersensitivity reactions when tested with soluble canine testicular extracts. Reactions did not occur in normal dogs. Dogs with testicular atrophy had the most severe skin test responses. Seemingly, isoimmune responses to sperm antigens are involved in infertility caused by B canis infection of male dogs.     

First messenger regulation of mammalian sperm function via adenylyl cyclase/cAMP

When released into an appropriate environment, mammalian spermatozoa begin to capacitate and then continue until fully capacitated and able to fertilize. During capacitation in vitro, some cells 'over-capacitate' and undergo spontaneous acrosome reactions; this would be highly undesirable in vivo since already acrosome-reacted spermatozoa are non-fertilizing. Recent studies have revealed that seminal plasma contains several small molecules that bind to specific receptors on the sperm plasma membrane and act as 'first messengers', causing biologically important changes in availability of the 'second messenger' cAMP. Fertilization promoting peptide (FPP), calcitonin and adenosine all regulate cAMP production, stimulating it in uncapacitated spermatozoa and then inhibiting it in capacitated cells; in contrast, angiotensin II stimulates cAMP throughout capacitation. The molecules that regulate cAMP appear to do so via G protein-modulated changes in membrane associated adenylyl cyclases (mACs). Both mouse and human spermatozoa have been shown to have Galphas and Galphai2, as well as several isoforms of mAC, located in the same regions as the specific receptors. Thus spermatozoa possess the required elements for several separate signal transduction pathways, many of which regulate mAC/cAMP and so maintain sperm fertilizing ability. In vivo, such responses could increase the chances of successful fertilization.     

猪精子获能前后细胞亚组分蛋白分离及酪氨酸磷酸化蛋白的鉴定

本研究对猪精子获能前后细胞亚组分蛋白进行分离以及对酪氨酸磷酸化蛋白进行鉴定,旨在为哺乳动物精子受精生物学研究奠定理论基础。利用动物精子体外获能培养、细胞亚组分分离技术及蛋白免疫印迹的方法,分离猪精子细胞亚组分蛋白及酪氨酸磷酸化蛋白鉴定。结果表明,猪精子经过获能培养后各项活力指标均得到显著提高,且与精子蛋白发生酪氨酸磷酸化修饰密切相关;获能精子中126、108、79ku的高分子量蛋白磷酸化程度明显高于未获能精子;分子质量约为25、47、50ku的膜蛋白及47ku胞浆蛋白发生酪氨酸磷酸化,其中25、47ku的膜蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05);分子量约为23、37、42～50ku的核蛋白发生酪氨酸磷酸化,获能精子中23ku的核蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05)。结果提示,猪精子细胞不同亚组分中,发生酪氨酸磷酸化修饰的蛋白以膜蛋白及核蛋白为主,同时有少量的胞浆蛋白。     

Biochemical observations on beagle dog semen.

Semen samples were collected at weekly intervals for six weeks from eight sexually mature beagles previously shown to produce normal ejaculates. Seminal plasma and sperm fractions were separated by centrifugation and the sodium, potassium, alanine and aspartate aminotransferases, acid and alkaline phosphatase concentrations in the two fractions determined. Regression analysis of the mean weekly values obtained from physical and biochemical examination of the ejaculates showed that sodium ion concentration was highest in seminal plasma. The highest levels of aminotransferases were found in sperm fractions. Those enzymes may be indices of abnormal or damaged spermatozoa. Acid and alkaline phosphatase activity was 100 times greater in seminal plasma than in sperm fractions. Phosphatase concentrations are likely to be dependent on prostate activity. Measurement of acid phosphatase in canine semen therefore may be a useful index of prostate function. The motility of the semen samples was independent of the potassium concentration in seminal plasma. However, there was some evidence of a correlation between sperm motility and the enzyme and sodium content of seminal plasma.