精子载体转基因技术的最新研究进展

用动物精子作为外源DNA载体已成为转基因的主要方法之一,并在多种动物上获得成功.最近,人们对外源DNA与精子结合的分子机理进行深入研究发现外源DNA可与精子膜上30～35 kDa蛋白质特异结合,结合强度受精子膜上MHCII因子的调节.精清中的IF-1因子能抑制精子结合外源DNA,从而维持物种的遗传稳定性.精子结合外源DNA的量是恒定的,部分DNA能进入精子核内,CD4蛋白具有转运外源DNA进入精子核内的功能.进入核内的外源DNA牢固结合在精子核骨架上,然后整合到染色体的特异位点.但是,精子与外源DNA结合后能激活精子内的核酸酶,进而使外源DNA分子降解,可能是导致这种转基因方法稳定性差的主要原因.     

Sperm storage and low incidence of multiple paternity in the European pond turtle, Emys orbicularis: A secure but costly strategy?

The freshwater pond turtle, Emys orbicularis, has recently suffered from population declines throughout its range, mainly due to habitat destruction. The mating strategies of this species were studied using genetic data from successive clutches within and between years. To test for the occurrence and frequency of multiple paternity and sperm storage, genetic paternity at six microsatellite markers was assessed in 114 embryos and hatchlings from single and subsequent clutches of 11 females (including clutches from the same or consecutive years). Multiple paternity was rare and only found in two out of 20 clutches from 11 females. All annual successive clutches and 58% of the clutches in the next year, were fertilized with sperm from the same male. The use of stored sperm is thus a frequent strategy in E. orbicularis. However, hatching rate, hatchling mass, and hatchling length decreased in clutches fertilized by stored sperm, suggesting sperm depletion or deterioration through time. The occurrence of stored sperm despite an associated reduced reproductive output indicated that mating and/or the fertilization process is costly to females. The low incidence of multiple paternity may simply be the residual consequence of the capacity to store viable sperm. These results provide important and innovative insights for the conservation of E. orbicularis. In threatened populations, management strategies may aim to enhance effective copulations in order to increase the reproductive output of females.     

Sperm winding in Collembola

The winding process leading to the formation of rolled-up spermatozoa in Collembola is described. It is a phenomenon common to all collembolan species examined so far and it starts with the organization of a cytoplasmic flattened evagination beneath the head of the elongated spermatid, which gives rise to an extracellular cavity. Within this cavity secretory material produced by the epithelial cells of testes is progressively stored. The spermatozoon, whose cytoplasm is reduced to a thin layer, enrolls around this material. The final appearance is that of a spheroidal or lenticular structure containing dense material in the central extracellular cavity; this cavity is surrounded by the flattened sperm in whose cytoplasm the sperm components forms several spires. The material into the extracellular cavity becomes progressively denser and has a different appearance in the species examined.     

海藻糖和甘油相互协同提高绒山羊精液冷冻保存品质

本实验旨在研究不同浓度的海藻糖和甘油相互协同对绒山羊精液冷冻保存的影响。将0 mmol/L (T0)、50mmol/L(T50)的海藻糖和0%(G0)、1%(G1)、2%(G2)、3%(G3)的甘油分别组合为T0G0、T0G1、T0G2、T0G3、T50G0、T50G1、T50G2和T50G3。以Tris-柠檬酸-葡萄糖(TCG)为基础稀释液,分析不同组合对绒山羊精液冷冻保存的影响。检测解冻后精子活力、运动参数、DNA完整率、顶体、质膜完整率、抗氧化水平。结果表明:海藻糖和甘油联合添加时精子冷冻保存效果显著高于单独添加海藻糖或甘油组,其中T50G1组作用最显著。在基础稀释液中添加50 mmol/L(T50)的海藻糖时,精子冷冻解冻后的活力、质膜完整率随着甘油浓度的升高而降低,而在基础稀释液中不添加海藻糖时,精子冷冻解冻后的活力、质膜完整率随着甘油浓度的升高而升高。综上表明,海藻糖和甘油通过协同作用提高精子冷冻保存效果,两者发挥作用的最适浓度为50 mmol/L海藻糖和1%甘油,并且海藻糖和甘油协同作用对精子活力以及质膜完整性具有浓度依赖性。     

雄性生殖细胞介导外源DNA转移的新方法

介绍并评价在传统精子载体法的基础上的改进与发展,即辅以生精细胞显微授精的基因转移;输精管注射法体内转染成熟精子和曲细精管注射法转染生精干细胞.这些方法的应用将为哺乳动物基因转移提供更为方便和高效的技术手段.     

Transfer of MicroRNAs From Epididymal Epithelium to Equine Spermatozoa

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.     

GnRH和LHR基因多态性和牛精液品质性状的相关分析

本试验采用PCR—RFLP方法分析了GnRH基因外显子2和LHR基因内含子9在144头中国荷斯坦牛和79头河南地方肉牛品种中的多态性,利用最小二乘法分析多态位点不同基因型与精液品质性状的关系。研究结果表明,2～3岁荷斯坦牛的鲜精活力显著高于4岁以上的牛,而畸形率显著低于7岁以上的牛。对2～4岁荷斯坦牛不同精液品质性状的简单相关分析表明,畸形率与顶体完整率和冻精活力呈显著的负相关（相关系数r分别为-0．736和-0．500）。不同基因型与精液品质性状的关联分析结果表明,144头中国荷斯坦牛所研究位点不同基因型对精液品质性状没有显著影响。而河南地方肉牛GnRH基因外显子2的A883G位点GG基因型的精子密度显著低于AA和AG基因型．LHR基因G51656T位点的TT基因型精子密度显著高于GT基因型,未检测到GG基因型。并且发现随着年龄的增长,种公牛的精液品质逐渐变差。GnRH和LHR基因可作为影u向肉牛精液品质性状的候选基因。     

不同稀释剂对猪冷冻精液的影响

手握法采集12月龄大约克种公猪精液,在37℃下离心,弃去精清,收集浓缩段富含精子部分,用7种不同的稀释液进行稀释,应用液氮熏蒸法制作颗粒冻精。结果表明,在7种稀释液中,7号稀释液优于其他6种稀释液(p<0.05);与其他的冷冻保护剂相比,甘油的冷冻保护性能较好(p<0.01),其适宜浓度为2~4ml/l;干解冻(40~45℃)效果优于湿解冻,解冻后精子活率达0.46~0.49(p<0.05);稀释液中添加安钠咖可有效地延长精子的冻后存活时间(p<0.01)。     

附睾对精子表面抗原修饰作用的亲合细胞化学研究

为了认识附睾对精子表面抗原修饰作用的分子机制,本文应用HRP—WGA、HRP—ConA、HRP—SBA、HRP—RCA—Ⅰ和HRP—UEA—Ⅰ分别对昆明小鼠附睾头、附睾体与附睾尾切片进行了亲合细胞化学研究,并用HRP—WGA、HRP—ConA和HRP—SBA分别对取自附睾头和附睾尾的精子涂片进行了标记,以明确附睾管糖复合物分泌功能与精子表面抗原变化之间的关系。WGA对附睾内排精管上皮细胞的标记很不均一,直至附睾尾部,上皮细胞才呈均一的强阳性反应。从附睾头至附睾尾ConA对排精管上皮细胞的标记由强变弱,个另细胞甚至完全不被标记。SBA对各段排精管上皮细胞的标记基本相同。RCA—Ⅰ对排精管上皮细胞的标记主要发生在静纤毛上,并呈强阳性反应,且各段上皮细胞的标记反应相似。UEA—Ⅰ对附睾头和附睾体内排精管上皮细胞的静纤毛呈强阳性标记,且在顶端胞质内有一强阳性颗粒,在附睾尾段,细胞内强阳性颗粒消失,静纤毛标记强度也减弱。WGA对涂片上精子的标记反应相近。ConA在精子顶体区的标记强度随着精子向附睾尾的推移而略增。与此相反,SBA在精子顶体区的标记强度则随着精子向附睾尾的推移而减弱。上述结果表明,附睾排精管上皮细胞具有分泌多种糖复合物(主要是糖蛋白)的功能,并籍此功能实现其对精子表面抗原的修饰作用。     

大菱鲆精子低温短期保存

大菱鲆(Scophthalmusmaximus)精子仅能体外存活10min左右,根本来不及从养殖场带回实验室,而生产单位的实验条件又有限,这严重限制了大菱鲆精子的实验研究。本文利用TS19做稀释液,6%DMSO作抗冻剂,在4℃下保存大菱鲆精子,每隔一段时间观察1次被保存精子的成活率。结果发现,大菱鲆精子体外存活时间可延长到3h。用保存3h的大菱鲆精子做授精实验,在各种精卵比的情况下,受精率与鲜精对照组没有显著差异。本实验保存大菱鲆精子的方法完全满足了大菱鲆精子短距离运输的要求。