受精蛋白β在人精子表面的免疫组织化学定位

以人受精蛋白β整联蛋白配体区为抗原免疫小鼠并制备单抗,应用免疫组化方法研究了受精蛋白β在不同状态人精子表面的分布规律。结果表明受精蛋白β散在分布于新鲜人精子的头部,获能后则向顶体区集中,顶体反应过程中同时出现表达量及分布位置的急剧变化,最后定位于精子头后部。其分布规律与动物受精蛋白有所不同。受精蛋白β在人精子表面分布的位置及其变化揭示其在受精过程中的重要作用。

哺乳动物精卵黏附和融合的分子基础

哺乳动物受精是一个多分子参与的多级过程,在精卵融合形成一个受精卵时达到顶点。新的研究已表明:精卵黏附后,精子膜从赤道区开始与卵膜融合;有精子膜上的去整合蛋白金属蛋白酶蛋白家族(ADAM)、Izumo、附睾蛋白DE、卵膜上的整合蛋白、四次跨膜蛋白CD9、CD81、糖基磷脂酰肌醇锚定蛋白等多种分子参与精卵黏附和融合过程。

精子膜抗原的研究新进展

精子表面的膜抗原是精子发生成熟过程中的重要标志分子 ,对其深入研究有助于揭示精子发生成熟及受精过程的生理机制和不育的病理改变。本文回顾了近年来随着分子生物学技术的广泛应用 ,传统的一些重要的精子膜抗原相继得到了克隆和测序 ,一些新的表达精子膜抗原的基因也陆续被发现 ,为从蛋白水平进一步深入研究其功能以及免疫避孕疫苗的筛选奠定了基础。

一种新的混合斑精子分离方法

制备了精子表面膜抗原受精素β蛋白（Fertilinβ）的特异性抗体,通过抗原-抗体反应和抗体固相偶联技术,将抗体连接到琼脂糖球珠上,建立了混合斑精子分离纯化的新方法.首先,通过聚合酶链式反应（PCR）扩增编码人受精素β蛋白第341~373位氨基酸的基因序列,构建PGEX-4T-1/Fertilinβ原核表达质粒;其次,诱导表达GST-Fertilinβ融合蛋白,制备受精素β蛋白多克隆抗体,免疫荧光检测结果表明,受精素β蛋白定位于精子的头后部,并且阴道上皮细胞没有受精素β蛋白的表达;最后,将受精素β抗体与ProteinA琼脂糖球珠连接构建固相抗体系统,将精子吸附于表面,可从混合斑（精子与阴道上皮细胞混合液）中分离纯化精子.本文为性犯罪案件侦破提供了新的方法.

Impact of asialoglycoprotein receptor deficiency on the development of liver injury

The asialoglycoprotein （ASGP） receptor is a wellcharacterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis （RME）. The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the missorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously, we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease （ALD）. The studies revealed profound ethanol- mediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations, studies were performed utilizing knockout mice （lacking a functional ASGP receptor） to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti- Fas （CD95） antibody, carbon tetrachloride （CCh） and lipopolysaccharide （LPS）/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels, histopathological scores, as well as hepatocellular death. Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxinmediated liver diseases.

Research on the Behavior and Egg Fertilization Rate of Dwarf Layers and White Leghorn Chickens Fed in Floor-Rearing System

Influenced by animal welfare requirements and expensive labor cost, producers tend to feed layers in non-cage system such as floor rearing. With the development of commercial lines of dwarf layers, the property of dwarf layers＇ natural mating is drawing more and more attention. In this study, the authors hybridized Nongda Ill dwarf layers with White Leghorn chickens in floor-rearing system to research whether dwarf layers had defect in natural mating. Two steps of experiments were performed. The authors detected the fertilization rate of collected eggs, observed chickens＇ behavior in step I when a hen from other group was suddenly put into and recorded copulation number of every group in step II from 7：30 to 19：30. The results indicated that drawf cocks were as good as Leghorn cocks in natrual mating under floor-rearing system while drawf hens were better than Leghorn hens. Observation results showed no difference on reaction to intermixed hens between drawf layers and Leghorn layers. The best proportion of male to female for drawf layers was 1：8 in this experiment and dwarf cocks tended to mate dwarf hens rather than Leghorn hens.

A Protein Extracted from Mouse Sperm That Plays an Important Role in Fertilization

A membrane protein was isolated from mouse sperm heads that had undergone acrosomal reaction induced by C2+ ionophore, A 23187, which, with a molecular weight of 77.6 kd, shows capability to block egg-sperm fusion. As revealed by analysis usintg isotopic markers, this protein is one of the chief membrane proteins of inner acrosomal membrane or the outer membrane of equatorial segment and Post-acrosomal region; treatment of mouse sperms with 0.6 μg/ml of the Purified protein for 30 minutes reduced the sperm-egg fusion index by 51%.The above results led us to the conclusion that the protein is an active participant in sperm-egg fusion. The possible existence of sperm receptor on egg plasma membrane was discussed.

Actin,more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig(1876),enormous progress and insights have been made on this topic.However,the precise molecular mechanisms underlying fertilization are largely unknown.The two most dramatic changes taking place in the zygote immediately after fertilization are:(i) a sharp increase of intracellular Ca2+ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave,and(ii) the concomitant dynamic rearrangement of the actin cytoskeleton.Traditionally,this has been studied most extensively in the sea urchin eggs,but another echinoderm,starfish,whose eggs are much bigger and transparent,has facilitated experimental approaches using microinjection and fluorescent imaging methodologies.Thus in starfish,it has been shown that the sperm-induced Ca2+ increase in the fertilized egg can be recapitulated by several Ca2+ -evoking second messengers,namely inositol 1,4,5-trisphosphate(InsP3) ,cyclic ADP-ribose(cADPr) and nicotinic acid adenine dinucleotide phosphate(NAADP) ,which may play distinct roles in the generation and propagation of the Ca2+ waves.Interestingly,it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca2+ signaling.As it is well known that Ca2+ regulates the structure of the actin cytoskeleton,our finding that Ca2+ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca2+ signaling may form a'positive feedback loop'that accelerates the downstream events of fertilization.Perturbation of the cortical actin networks also inhibits cortical granules exocytosis.Polymerizing actin bundles also compose the'acrosome process,'a tubular structure protruding from the head of fertilizing sperm. Hence,actin,which is one of the most strictly conserved proteins in eukaryotes,modulates almost all major aspects of fertilization.

AURKB and MAPK involvement in the regulation of the early stages of mouse zygote development

Aurora kinases have become a hot topic for research as they have been found to play an important role in various stages of mitotic cell division and to participate in malignant conversions of tumors. The participation of Aurora kinases in the regulation of oocyte meiosis has been recently reported, but their participation in mammalian early embryonic development remained unclear. The object of our study was to establish the spatio-temporal expression pattern of Aurora kinase B (AURKB) in mouse zygotes during the first cleavage, to reveal its functions in the early development of mouse zygotes, and to define the involvement of AURKB in mitogen-activated protein kinase (MAPK) signaling. Our results showed that in mouse zygotes AURKB expression increased in G1 phase and peaked in M phase. AURKB protein distribution was found to be in association with nuclei and distributed throughout the cytoplasm in a cell cycle-dependent manner. Functional disruption of AURKB resulted in abnormal division phenotypes or mitotic impairments. U0126, a specific mitogen-activated protein kinase kinase (MEK) inhibitor, caused significantly altered morphologies of early embryos together with a decrease in protein expression and kinase activity of AURKB. Our results indicated that the activity of AURKB was required for regulating multiple stages of mitotic progression in the early development of mouse zygotes and was correlated with the activation of the MAPK pathway.