蕨精子单克隆抗体制备Ⅰ：确定精子抗原性

蕨精子单克隆抗体制备Ⅰ：确定精子抗原性方瑾,刘宁,周云龙,钱晓菁,陈令静（北京师范大学生物系,北京１０００８７５）梁华,鞠振林,潘香刚（中国农业科学院生物技术中心工试基地,北京１００８１）寻找和研究配子的特异性蛋白对于研究配子识别,深刻认识高等植物受...     

水蕨精子器及精子形态的显微观察

水蕨(Ceratopteris thalictroides)隶属于水蕨科(Cer- atopteridaceae)水蕨属(Ceratopteris),为一年生多汁淡水生草本蕨类。采集水蕨成熟孢子,按常规方法将其接种到改良knop's液体培养基中,置于人工培养箱中培养。培养条件为全天光照,光照强度约2000 lx;湿度约为60%～70%。     

水蕨精子器及精子形态的显微观察

研究人员在1月出版的《自然-神经科学》中报告说,人在睡觉时,其清醒时的经历会重复出现在大脑的几个部位。     

精子因素对精子载体法制备转基因山羊的影响

精子具有主动结合、转运、整合外源DNA的能力,并在受精时导入卵母细胞,获得转基因动物.精子介导基因转移(sperm-mediated gene transfer,SMGT)是目前获得转基因动物简单而高效的方法之一.精子因素是影响SMGT方法生产转基因动物的重要方面.本论文结合我们的研究针对转染用山羊(Capra hircus)精液的来源、精子质膜完整性、精液品质及发育阶段等精子因素影响精子结合外源DNA和SMGT方法生产转基因山羊的效率进行了论述,并从这些影响因素入手,提出了筛选精子供体、保持精液品质、调控质膜等措施,提高精子转染外源DNA能力和生产转基因动物的效率.     

精子鞭毛轴丝结构相关基因异常与弱精子症

精子鞭毛轴丝是精子运动的主要动力来源,参与鞭毛组装和运动调控的基因变异可导致精子活力降低,从而引起弱精子症(asthenozoospermia, ASZ)。常见的弱精子症包括两大类:(1)精子鞭毛在光学显微镜下无明显畸形,(2)精子鞭毛多发形态异常(multiple morphological abnormalities of the sperm flagella, MMAF)。弱精子症主要由轴丝组分编码基因变异所致,在过去的十年里,在揭示致病基因方面取得了显著进展。在MMAF的遗传研究领域,中国和法国是两个涉及比较广的国家。通过系统文献检索和Meta分析中国和法国关于MMAF的基因变异研究,纳入1 796名不育男性参与者,结果表明,在中国的弱精子症患者中, DNAH1基因的突变比例显著高于法国(OR=4.97,95%CI=[1.70; 14.49], P<0.01)。而CFAP43、CFAP44、CFAP251等基因在两国间未显示显著性差异(P>0.05)。这一发现为理解弱精子症的遗传变异的多样性奠定了基础。     

大鲵精子结构研究

为了解大鲵精子超微结构,应用扫描电镜和透射电镜开展了大鲵精子形态结构研究。结果显示:大鲵精子由头部、颈部和尾部3部分组成。精子总长216.36μm±9.93μm(n=30),头部长65.80μm±3.70μm(n=30),颈部较短,多不明显,尾部长153.52μm±3.22μm(n=30)。头部由顶体、穿孔器和细胞核组成;颈部包括核窝、近端中心粒及远端中心粒、线粒体、轴丝和轴纤维;尾部无明显分段,由轴丝、轴纤维、轴丝旁纤维和波动膜组成。大鲵精子内线粒体较少,可能与精子运动缓慢、精子活力维持时间短有关;成熟过程中精子细胞头部包围的胞质分泌物中含有一定数量的线粒体。     

大黄鱼精子的超微结构

大黄鱼的精子由头产和尾部两部分组成。头部结构较为独特,其腹侧有一较大的细胞核,背部有中心粒复合体。头部的后端是袖套。细胞核的腹面稍向外突出背面则稍向内凹。细胞核中的染以质浓缩成致密的团块状。团块状的染色质之间分布着松散的纤维状染色质。植入窝位于细胞核的背部表面,由细胞核背面向内凹陷而成,呈一沟状,其走向与精子的长轴平行。     

羊精子体外获能

本文发展了一种羊精子体外获能培养基——mTs或RSCM。羊精子在该培养基中(39℃.pH7.8和5％ CO_2,95％空气)预先培养7小时,可使大部分精子获能。获能精子呈现超激活运动,并可穿透去透明带仓鼠卵,穿透率分别为78.5±14.3％和96.7±2.3％,这种作用可被同种精浆逆转。获能精子与同种卵的受精率为83.3％。16个受精卵等量移入2只假孕兔输卵管壶腹部中。72小时后回收到12个胚胎。其中6个胚胎已发育为4—8细胞阶段,将这些细胞等量移入2只受体母羊输卵管壶腹部。其中1只妊娠,并维持到2个月之久。     

自然界最长的精子

夏天，人们容易受到蚊子叮咬，但为什么蚊子对有些人总是“敬而远之”？英国科学家在一项对人体汗液的研究中，发现了其中可以驱赶蚊子的化学物质，这些化学物质在每个人的汗液中都会自然产生，但不被蚊子叮咬的人的汗液中这些化学物质的含量比别人高。     

精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。     

影响山羊体外受精的因素

以屠宰山羊卵母细胞为材料研究了公羊个体、附睾不同部位精子、成熟培养和受精时卵丘存在与否、卵丘扩展程度及卵龄对山羊体外受精的影响。结果表明 :1)不同公羊精液在受精、卵裂和桑椹 /囊胚率上都有显著差异 ;2 )附睾尾精子和鲜精的受精、卵裂和桑椹 /囊胚率无显著差异 ,但显著高于附睾体和附睾头精子 ;3)成熟培养 2 4和 2 7h卵母细胞的的桑椹胚 /囊胚率显著高于培养 2 1和 30h卵母细胞 ;4 )卵丘扩展 3和 4级卵母细胞受精和桑椹胚 /囊胚率显著高于扩展 0和 1级卵母细胞 ;5 )成熟培养前机械去卵丘严重影响卵母细胞体外受精和桑椹胚 /囊胚率 ;6 )受精前完全去掉卵丘显著影响桑椹胚 /囊胚率     

Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.     

Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach

Intracytoplasmic sperm injection (ICSI) is advantageous when only very few spermatozoa are available for insemination. Bovine spermatozoa were injected individually into matured oocytes using a piezo electric actuator. Spermatozoa were "immobilized", by scoring their tails immediately before injection, or "killed", by repeated freezing and thawing. About 4 h after ICSI, the oocytes with two polar bodies (activated by sperm injection) were selected and treated 5 min with 7% ethanol before further culture. When examined 19-21 h after ICSI, nearly 90% of the oocytes were fertilized normally (two pronuclei and two polar bodies) irrespective of the sperm treatment (immobilization or killing) prior to ICSI, but subsequent preimplantation embryo development was much superior (cleavage 72%: blastocysts 20%) after ICSI with immobilized spermatozoa than by using killed spermatozoa (cleavage 28%; blastocysts 1%). Ethanol activation of bovine oocytes with two polar bodies 4 h after ICSI improved the cleavage (33% versus 72%) and blastocyst (12% versus 20%) rates markedly (P < 0.05). Five normal calves were born after transplantation of ten blastocysts to ten surrogate cows. These results show that piezo-ICSI using immobilized spermatozoa, combined with ethanol treatment of sperm-injected oocytes, is an effective method to produce bovine offspring.     

Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa

Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the ICSI ova cleaved after treatment with thimerosal. Ionomycin activation after 24 and 30 h of oocyte maturation resulted in 29 and 48% cleavage rates, respectively. Ionomycin combined with DMAP resulted in 49, 6 and 3% cleavage, morula and blastocyst rates, respectively, when oocytes were activated after 24 h maturation. In Experiment 3, rates of cleavage (45-60%) and development to morulae (4-13%) and blastocysts (1-5%) stages following ICSI were not different (P>0.05) among three stallions. Treatment of stallion spermatozoa with ionomycin did not affect cleavage or development of ova fertilized by ICSI. The chromosomal constitution of blastocysts derived from ICSI was bovine, not bovine and equine hybrids. In Experiment 4, to make male and FPN form synchronously, colchicine and DMAP were used for 4 h to inhibit oocytes at metaphase during activation; 63% of oocytes were still at metaphase 8h after ICSI when treated with colchicine, and 50% of sperm nuclei were decondensed. About 18 h after ICSI, 21 and 50% male and FPN had formed, respectively, but cleavage rates were low, and only 1% developed to morulae. In Experiment 5, to test if capacitated equine sperm could fuse with the bovine oolemma, capacitated spermatozoa were injected subzonally (SUZI). Of the 182 SUZI oocytes, 49 (27%) contained extruded second polar bodies. After activation of oocytes with second polar bodies, 44, 22 and 15% developed to 2-, 4- and 8-cell stages, respectively, but development stopped at the 8-cell stage. None of the unactivated oocytes cleaved. In conclusion, equine spermatozoa can decondense and form MPN in bovine oocytes after ICSI, but subsequent embryonic development is parthenogenetic with only bovine chromosomes being found.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm

The present study investigated the correlation of sperm movement in the ooplasm, pretreatment of sperm with dithiothreitol (DTT) and sperm freezing with the development of porcine embryos derived from modified intracytoplasmic sperm injection (ICSI). In vitro, matured gilt oocytes without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail-first. In Exp. 1, frozen-thawed sperm were categorized into three groups: impaired, immotile or motile. Oocytes injected with motile sperm (43.6%) showed a higher (P < 0.05) fertilization rate compared to oocytes injected with impaired or immotile sperm (34.5 or 37.2%). The survival rate was significantly higher (P < 0.05) in oocytes injected with impaired sperm (92.9%) than in oocytes injected with immotile or motile sperm (84.8 or 86.7%). No differences were observed in the rates of cleavage or blastocyst formation, and in total cell number of blastocysts among three groups of oocytes. In Exp. 2, motile frozen-thawed sperm were pretreated with DTT before injection and non-treated sperm served as controls. Higher rates (P < 0.05) of fertilization, male pronucleus (MPN) and decondensed sperm head (DSH) formation were observed in oocytes injected with control sperm (41.1, 50.0 and 91.1%, respectively) than in oocytes injected with DTT-treated sperm (22.1, 30.2 and 72.1%, respectively). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In Exp. 3, motile frozen-thawed or fresh sperm without DTT pretreatment were injected into oocytes. The rates of fertilization and MPN formation were significantly higher (P < 0.05) in oocytes injected with fresh sperm (59.8 and 73.5%) than in oocytes injected with frozen-thawed sperm (36.7 and 59.2%). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In conclusion, the present study clearly demonstrated that sperm movement in the ooplasm, use of DTT and fresh spermatozoa did not significantly affect on embryo development in porcine modified ICSI.     

Improved fertilization and embryo development resulting in birth of live piglets after intracytoplasmic sperm injection and in vitro culture in a cysteine-supplemented medium

The effects of cysteine treatment on fertilization rate, intracellular concentration of glutathione, and embryo development in vitro and after embryo transfer were examined following intracytoplasmic sperm injection (ICSI) of in vitro-matured porcine oocytes using a piezo drive unit. Culture of presumed zygotes after ICSI with 1.71-3.71 mM cysteine for 3-12h improved (P<0.05) fertilization rates as compared to treatment with 0.57 mM cysteine or to controls (0mM) (56 to 68%, 48%, 35%, respectively). Extension of treatment time with cysteine beyond 3h did not further increase fertilization rates, suggesting that cysteine promoted early developmental events after ICSI (e.g. decondensation of sperm chromatin). There was no effect of cysteine supplementation on oocyte glutathione levels after ICSI. Pretreatment of spermatozoa for 3h with 1.71 mM cysteine did not improve fertilization rates. The incidence of blastocysts formation when cultured in 1.71 mM cysteine for 3h after ICSI was 31%, which was higher (P<0.05) than controls (18%). Transfer of 20-38 embryos cultured with 1.71 mM cysteine for 3h after ICSI to each of seven recipients yielded three deliveries with an average litter size of 4.0. We concluded that cysteine supplementation for the first 3h after ICSI improved fertilization and embryo development rates, with no influence on glutathione levels in oocytes, and that the cysteine-treated ICSI embryos developed to full term. The study also showed that porcine oocytes matured in a chemically defined medium had the ability for full-term development after piezo-ICSI without additional treatments for oocyte activation.     

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.