奶牛Y精子膜蛋白的提取与分析

本实验旨在对奶牛Y精子膜蛋白的提取与SDS-PAGE分析,是分离纯化奶牛Y精子特异膜蛋白基础与关键。本实验采用超声波破碎法将精子膜与精子分离,分离后的精子膜粗品采用蔗糖密度梯度离心法进行纯化。纯化的精子膜经膜蛋白提取液(去污剂)提取,透析浓缩。经SDS-PAGE银盐染色,结果表明该方法成功地提取到膜蛋白,得到膜蛋白电泳图谱。BIO-RAD分析得知Y精子膜蛋白种类至少有10种,分子量范围为7.94kD～166.65kD,其中尤以15kD的蛋白含量最多。奶牛Y精子膜蛋白的提取与鉴定为深入研究Y精子特异膜蛋白在受精过程中的作用,从而获得一种更经济、更安全、更有效的奶牛性别控制方法提供了实验依据。     

精子性染色体相关基因转录本及检测方法研究进展

精子形成过程中,基因表达经历了减数分裂性染色体失活,但有很多基因在减数分裂后期转录又重新被激活。转录产物研究表明,X和Y染色体特异基因在不同物种精子中都发生了不同程度的转录,并且对转录产物的研究方法也取得了巨大进展,产生了微阵列、SAGE及抑制消减杂交等先进的技术手段,随着这些技术的日益成熟和完善,通过深入研究,精子中性别相关基因转录表达调控机理将越来越清晰,并推动性别特异蛋白的研究取得更大进展。     

重组于脂双层的人精子质膜Ca2+通道的膜片箝研究

跨膜离子流动在启动配子间的相互作用,如顶体反应过程中有重要作用。．但由于精子太小,无用论细胞内记录或膜片箝技术都难以对哺乳动物精子膜的离子通病进行研究。本工作将通道蛋白重组于脂双层,在电压箝位下记录了人精子膜的Ｃａ＾２＋通道电流。由１２个健康人的精子分离的膜蛋白通过融合重组于脂双层后,在ＣａＣｌ２溶液中（ｃｉｓ５０／／ｔｒａｎｓ １０ｍｍｏｌ／Ｌ）记录到两类单通道活动,它们的平衡电位都接近Ｃａ＾２     

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。     

优雅蝈螽精子形成期间微丝和微管蛋白的动态变化

【目的】分析微丝和微管蛋白在优雅蝈螽Gampsocleis gratiosa精子形成过程中的作用,为昆虫精子顶体复合体形成和细胞核重建机制研究奠定基础。【方法】应用免疫荧光、PAS-苏木精染色和透射电镜等方法,对优雅蝈螽成虫的精巢、雄性贮精囊和雌性受精囊内精子的发育以及微丝和微管蛋白在精子形成各个时期的分布进行了观察。【结果】精巢中,早期圆形精子细胞中微丝在精子细胞的某区域大量聚集,而微管蛋白随机分布在细胞质中。伸长的精子细胞中,顶体开始形成时,微丝首先在亚顶体区域出现,历经球形、短棒形,然后向细胞核的两侧扩展成倒"Y"形,接着形成箭头形;在顶体的外周即微丝的周围,细胞核周围以及鞭毛中发现微管蛋白。在雄虫贮精囊和雌虫受精囊中,精子和精子束中仅有微管存在,且仅存在于鞭毛中;精子头部的微丝和微管蛋白均消失。【结论】综合分析,我们认为微丝和微管作为"脚手架"结构在优雅蝈螽精子形成期间参与顶体复合体形成和细胞核重建,精子成熟形成精子束过程中"脚手架"结构拆除。     

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     

精子膜麦芽凝集素结合糖蛋白抗原某些特性的研究

应用自制的抗牛精子膜麦芽凝集素结合糖蛋白血清,对兔、人、小鼠和仓鼠精子进行了免疫细胞化学定位,结果各种动物精子均呈阳性反应,且以精子顶体区标记最强,与麦芽凝集素亲和细胞化学的标记结果相似。用抗血清处理地鼠精子,再与同种卵子进行体外结合试验,结果精子与卵于透明带的结合受到显著抑制、本实验的结果提示,牛精子膜麦芽凝集素结合糖蛋白抗原具有种间交叉反应性,并可能在精子与卵子透明带结合过程中具有重要作用。     

精子形成期基因转录表达的研究进展

减数分裂后, 圆形精子细胞经过一系列变态过程最终发育为成熟精子。期间, 精子细胞质逐渐丢失, 其染色质组蛋白逐渐经过渡蛋白替换为鱼精蛋白, 染色质被致密包装并高度浓缩。很多学者认为, 精子转录活性被关闭, 不存在RNA。但近些年却在精子中检测到了种类繁多的转录本, 包括精子染色质重新包装所需蛋白的转录本及一些小分子RNA等。由于精子核内组蛋白没有完全被鱼精蛋白替换, 且染色质上包含一些核酸活性敏感位点, 推测精子存在一定的转录活性, 并通过激素和表观遗传修饰等调控转录。精子中的这些RNA一部分是精子形成过程中残留下来的, 另一部分是精子细胞适时表达的。深入研究精子形成中的基因转录表达, 可增进对精子形成与成熟遗传本质的理解, 为高效利用雄性配子进行生殖控制提供理论依据。文章综述了近年来精子形成期基因转录表达的研究进展, 并提出了未来的研究方向。     

正常精子和圆头精子差异表达蛋白的分离与鉴定

圆头精子症是一种表现为顶体缺失的男性不育症.为了研究正常精子和圆头精子的蛋白质组成差异,用固相pH梯度双向凝胶电泳和质谱分析等蛋白质组学方法,分离了30份正常和3份圆头精子标本的蛋白质.对其中16个在正常精子中有高丰度表达而在圆头精子中缺失,和1个在圆头精子中表达明显下调的蛋白质点,以及在圆头精子中存在而在正常精子中缺失的蛋白质簇W和点X,进行了肽质指纹分析和蛋白质鉴定,获得8个点的肽质量指纹图,经MS-Fit软件搜索SWISS-PROT数据库来鉴定其身分.发现其中3个点与高尔基体相关、2个点与蛋白酶体相关、2个点为锌指蛋白,对其功能和与圆头精子形成的可能关系进行了初步探讨.     

Dynamics of F-actin during spermiogenesis in Gampsocleis gratiosa ( Orthoptera: Tettigoniidae)

为阐明F-肌动蛋白在优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl精子形成过程中的动态变化,本研究利用微分干涉相衬技术和免疫荧光技术首次对优雅蝈螽精子形成过程中的F-肌动蛋白进行了细胞定位,利用透射电镜技术从超微水平观察了优雅蝈螽精子顶体复合体的结构.结果显示:精子形成早期,F-肌动蛋白富集于亚顶体区域,形态由“球状”转变为“棒锥状”;精子形成中期,F-肌动蛋白呈“倒Y型”分布于亚顶体区域和细胞核前端两侧;精子形成后期,亚顶体区域的F-肌动蛋白解聚消失,F-肌动蛋白呈“箭头状”,仅分布于顶体复合体扩张的两翼中.F-肌动蛋白动态变化伴随着细胞核和精子头部的形态改变,F-肌动蛋白的动态装配在精子顶体复合体形态构建和细胞核的形变中起着重要的作用.本研究还发现未成熟的精子尾部有一些富含F-肌动蛋白的细胞质微滴,与精子形成过程中多余细胞质和细胞器的外排有关.F-肌动蛋白的动态变化研究为进一步阐明细胞骨架蛋白在昆虫精子形成过程中的功能和作用机制奠定了基础.     

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.     

哺乳动物精子分离方法的研究进展

分离X、Y精子,用于人工授精或体外受精,是目前实现哺乳动物性别控制的最有效手段。本文对哺乳动物精子分离及分离精子纯度评估方法的研究历史及现状作一回顾和总结。     

Regulatory RNAs and chromatin modification in dosage compensation: A continuous path from flies to humans?

Chromosomal sex determination is a widely distributed strategy in nature. In the most classic scenario, one sex is characterized by a homologue pair of sex chromosomes, while the other includes two morphologically and functionally distinct gonosomes. In mammalian diploid cells, the female is characterized by the presence of two identical X chromosomes, while the male features an XY pair, with the Y bearing the major genetic determinant of sex, i.e. the SRY gene. In other species, such as the fruitfly, sex is determined by the ratio of autosomes to X chromosomes. Regardless of the exact mechanism, however, all these animals would exhibit a sex-specific gene expression inequality, due to the different number of X chromosomes, a phenomenon inhibited by a series of genetic and epigenetic regulatory events described as "dosage compensation". Since adequate available data is currently restricted to worms, flies and mammals, while for other groups of animals, such as reptiles, fish and birds it is very limited, it is not yet clear whether this is an evolutionary conserved mechanism. However certain striking similarities have already been observed among evolutionary distant species, such as Drosophila melanogaster and Mus musculus. These mainly refer to a) the need for a counting mechanism, to determine the chromosomal content of the cell, i.e. the ratio of autosomes to gonosomes (a process well understood in flies, but still hypothesized in mammals), b) the implication of non-translated, sex-specific, regulatory RNAs (roX and Xist, respectively) as key elements in this process and the location of similar mediators in the Z chromosome of chicken c) the inclusion of a chromatin modification epigenetic final step, which ensures that gene expression remains stably regulated throughout the affected area of the gonosome. This review summarizes these points and proposes a possible role for comparative genetics, as they seem to constitute proof of maintained cell economy (by using the same basic regulatory elements in various different scenarios) throughout numerous centuries of evolutionary history.     

Purification and identification of sperm surface proteins and changes during epididymal maturation

Surface membrane proteins have a key role in the sequential interactions between spermatozoa and oocytes. The aim of this study was to characterize protein changes occurring during post-testicular differentiation using a new overall approach to study surface membrane proteins of spermatozoa. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed for this purpose. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes. Identification of several known proteins and of new proteins related to the process of epididymal maturation showed the reliability of this protocol for specific purification of a subproteome and identification of new sperm membrane proteins. This approach opens up a new area in the search for male fertility markers.     

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.     

Sperm separation attempts via the use of albumin gradients in rabbits

The objective of this study was to evaluate the sedimentation method using albumin gradients to determine whether it could help pre-select sex in rabbits. Of the 21 litters produced during natural mating, the sex ratio was 52.8% males which did not differ (P>0.05) from 55.3% males (nine litters) produced via AI from semen recovered after sedimentation through albumin gradients. The results obtained in this study are consistent with some but not all previous data. The quality of the recovered spermatozoa improved (P<0.05) when compared to those in the whole ejaculate. The sex ratio results obtained in this study suggest that sedimentation methods via the use of albumin gradients may not be effective for separating X and Y spermatozoa. Furthermore, actual sex ratio data should be stressed in all future studies with X and Y sperm separation, in addition to the $\text{in}$$\text{vitro}$ data generated.     

A visual screen of a GFP-fusion library identifies a new type of nuclear envelope membrane protein.

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.