阻止精子的成熟 开发新型男性避孕药

开发男性避孕药在世界人口争增地区是常见的对策。最近德国Schering公司等正在开发阻止精子成熟的药物。[编者按]     

蛋白质组学技术在精子蛋白研究中的应用

从蛋白质组学研究的技术手段、蛋白质组学在人类不育及精卵相互识别并结合的机理研究、免疫法开展男性避孕方法的研究及蛋白质组学研究方法在家畜繁殖环节中的应用等几个方面阐述了蛋白质组学在人类生殖及动物繁殖环节相关研究中的重要作用。说明蛋白质组学已经成为生命科学未来发展的主要分支之一,为揭示生命个体的蛋白质动态变化提供了技术手段和理论基础,并将在药物开发,生命活动机理研究等方面发挥巨大作用,也必将会在家畜繁殖学领域发挥其应有的作用。     

唇精子早期入卵观察

用扫描电镜对唇成熟卵子及早期精子入卵过程进行观察.结果 显示,唇成熟卵子在动物极中央有一深凹陷的表面光滑的精孔器,其外径2.512 μm,内径2.330 μm,精子直径1.567 μm.混匀的精卵刚遇水时,没有精子进入精孔器.受精后1 s,精孔器内出现精子.受精后5 s,组织切片显示,精子已经进入卵子内,并形成具有强烈抑制多精入卵作用的受精锥.受精后10 s,精子在精孔器前庭集结,尚未形成受精塞.受精后20 s,在精孔器内形成受精塞.受精塞没有阻塞精孔管,经分析它不是来源于皮层反应产物.受精塞形成后,可以吸附入卵的精子,这对多精入卵有积极的抑制作用;精子尾部在入卵过程中相互缠绕,这也是减少多精入卵的重要机制.受精后30 s, 受精塞和吸附的精子向精孔器外移动.受精后50 s, 受精塞和吸附的精子堵塞精孔器.受精后60 s, 受精塞吸附的精子开始解体,但是由于精孔管未封闭,还有精子通过精孔管进入到质膜.在人工受精过程中,卵子的单精受精屏障会因其周围精子密度大、精子与卵子距离短、精子运动速度快而被打破,从而导致这些卵子出现多精入卵的现象.受精后80 s, 精孔管仍然没有封闭,精孔器附近的精子明显出现活动能力的差异:精孔器外面的精子活动能力最强,精孔管旁边的精子活动能力较弱;精孔管外堆积的精子活性消失,受精塞吸附的精子已开始解体,经初步分析,这可能是进入其内的精子耗能有所差异的结果.受精后100 s,受精塞吸附的精子解体.     

小鼠精囊自身抗原的原核表达和纯化

目的：在原核细胞中表达小鼠精囊自身抗原（SVA）,并对表达产物进行鉴定和纯化。方法：提取小鼠附睾组织总RNA,RT-PCR获得SVA的cDNA,设计并合成特异引物序列,进一步扩增出不含信号肽的SVA编码序列,连入原核表达载体pET28a中,经酶切和测序鉴定正确的重组质粒转化大肠杆菌Rosetta（DE3）感受态细胞,IPTG诱导表达,Western印迹分析表达产物His-SVA,采用Ni-NTA纯化融合蛋白His-SVA。结果：原核表达获得融合6个组氨酸的SVA,用抗His单克隆抗体进行Western印迹鉴定,检测到相对分子质量约18×10^3的目的蛋白,与理论值一致;经Ni-NTA纯化获得较高纯度的His-SVA融合蛋白。结论：获得了在大肠杆菌中表达的小鼠附睾蛋白SVA,为后续研究其对小鼠生殖的影响奠定了基础。     

哺乳动物精子中的ZP3结合蛋白研究进展

哺乳动物卵透明带糖蛋白ZP3(zona pellucida3)是介导精卵初级结合、诱发精子发生顶体反应的关键分子。目前已在精子中发现多种ZP3结合蛋白。95kD酪氨酸激酶受体可能通过其酪氨酸激酶活性介导ZP3诱发的顶体反应。β—1,4—半乳糖基转移酶与ZP3的糖基结合后,通过激活下游信号分子诱发顶体反应。精子蛋白sp56可能介导了顶体反应期间顶体基质与ZP之间的相互作用。透明带粘附素(zonadhesin)也是在顶体反应发生之后才与ZP发生相互作用。这些精子蛋白介导的下游信号事件将是下一步研究的热点。     

pCR3．1—bvLDH-C4′的构建及在体内外的表达

精子特异性乳酸脱氢酶(LDH—C4)在精子的运动和存活等生理活动中起着重要的作用。研究表明,LDH—C4接种鼠、兔等能够降低动物的生育率。通过RT—PER方法从布氏田鼠睾丸总RNA中克隆出编码抗原决定簇的LDH—C4基因片断;采用T—A克隆的方法将其插入到pCR3．1载体中构建重组载体pCR3．1—bvLDH—C4′,测序结果表明克隆的基因片断与已知小鼠相应片断有83％的序列同源。通过质脂体法转染HeLa细胞,RT—PCR证实其可在mRNA水平有效表达;通过肌肉接种BAIB/c小鼠,RT-PER结果表明其在体内也可有效表达。     

绵羊胞内单精子注射技术

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.     

蟋蟀精子表面LCA及ConA结合糖复合物的分布变化

LCA and ConA-binding glycoconjugates on cricket (Teleogryllus emma) sperm surface were detected with fluorescence microscope after FITC labelling for better understanding of the distribution of glycoconjugates during spermatogenesis and spermiogenesis. FITC-LCA and FITC-ConA were bound on the spermatocytes, and their distribution changes in the process of spermiogenesis were observed .In the testis sperm, FITC-LCA and FITC-ConA were mainly bound on the head and neck region. That is different from the mark pattern of spermatophore sperm, in which the nucleus, neck region and front of the tail showed obvious fluorescence mark, especially the acrosome complex and neck region exhibited stronger mark. The mark patterns of FITC-LCA and FITC-ConA were similar,though the former was distinctly clearer than the latter. But a little difference still exists in both of them. For example in the ninth stage of spermatid, FITC-LCA mark is located on the spermatid head and neck region, and FITC-ConA mark on the spermatid head, neck and front of the tail region. When fixed germ cells were treated with PBS instead of lectin solution, or fixed cells were incubated with lectin solution, which have been treated with 0.1 mol/L specific sugar inhibitor, i.e.α-D-mannose for FITC-LCA and FITC-ConA, and α-D-glucose for FITC-ConA, no mark was observed on the cells. Those results indicate that FITC-LCA conjugated glycoconjugates has the α-D- mannose residue, and FITC-ConA conjugated glycoconjugates has the α-D-mannose and α-D-glucose residue. The investigations show that the changes in glycoconjugates distribution of cricket sperm is similar to those of other insects and mammals. The evidence exhibit that a common rule of the glycoconjugates distribution on the sperm surface is followed by most of animal sperm which may relate to the function of sperm physiology.     

精子特异性乳酸脱氢酶的免疫学特性及其应用

精子特异性乳酸脱氢酶特异地存在于鸟类和哺乳类动物的成熟睾丸和精子中,为精子的运动和存活提供能量,它是一种自身抗原,其天然抗体不与体细胞LDH同工酶发生交叉反应,用LDH－C4免疫小鼠或免疫等能够诱导免疫应答,导致生育率的降低,因此在人类避免和鼠害控制方面将有较好的应用前景。     

精子介导的转基因技术

精子介导的转基因技术是近十几年发展起来的一种新技术.从各个种的实验证明,精子有瞬间吸收外源DNA的能力.这一过程由一系列因子起重要调节作用.一种特异的DNA结合蛋白介导了外源DNA与精子的结合,同时精浆中存在一种因子起抑制两者结合的拮抗作用.CD4分子与外源基因的内化有关.内化的DNA可能与精子核支架结构(nuclear scaffold)结合,并整合或重排.但仍需要大量实验数据进一步证明是否产生真正可遗传的转基因后代,及如何提高转基因效率,以使这一方法得到普遍的推广及应用.