精子和卵母细胞质量控制对山羊卵胞质内精子注射(ICSI)受精的影响

以体外成熟卵母细胞为材料研究了精子来源及制动处理方法、卵母细胞质量及注射后激活等因素对山羊ICSI效果的影响.结果说明,附睾头、体和尾精子ICSI后的受精率、卵裂率和桑椹胚/囊胚发育率与射出的鲜精精子都没有明显差异(p＞0.05),但带下注射时附睾头和体精子的受精和发育率显著低于附睾尾和射精精子.在以4种不同方法致死的精子中,室温保存24h的死精子ICSI受精、卵裂和桑椹/囊胚率虽然低于对照组,但是明显高于其它方式致死的精子;5℃保存15天的死精子受精和发育效果最差.0.0005%Triton X-100处理精子的受精率、卵裂率和桑椹/囊胚率显著(p＜0.05)高于制动对照组、不制动对照组和其它浓度组.经高渗处理法检测质量好的卵母细胞ICSI受精和胚胎发育效果显著好于质量差的卵母细胞.与对照组相比,A23187和Ionomycin/6-DMAP激活处理均显著(p＜0.05)提高ICSI的受精率、卵裂率和桑椹/囊胚发育率.因此,精子在附睾内的成熟过程主要与其获得与卵质膜融合能力有关;精液保存方法对精子受精能力的损伤程度有很大差异;适当浓度的Triton X-100处理可模仿精子制动;卵母细胞质量是影响ICSI效果的重要因素;注射精子后激活卵母细胞能保证山羊ICSI的受精效果.     

不同卵龄小鼠卵母细胞激活和受精的比较研究

本实验比较了不同卵龄的小鼠卵母细胞受酒精人工刺激后的激活率和体外受精率,以探索卵母细胞激活和受精的机制。向NIH雌鼠腹腔注射孕马血清促性腺激素（PMSG）7．5单位,48小时后注射人绒毛膜促性激素（HCG）7．5单位,于不同时间杀小鼠,取卵母细胞与卵丘细胞的复合体（OCC）。从注射HCG后到取OCC的时间视为卵母细胞的卵龄。将OCC置于含8％酒精的M2中7分钟,再在16中培养5小时后,用0．3mg/mL的透明质酸酶去卵丘细胞。卵母细胞形成原核或速即卵裂为激活的标志,将OCC加入已获能的精子悬液中,5小时后将从卵丘细胞中释放出来的卵母细胞转移到M16中,将日发生卵裂为卵母细胞体外受精和激活的标志。小鼠卵母细胞卵龄为20h,其激活率为81．6％,速即卵裂率为48．0％;而卵龄进一步增加到24h,激活率和卵裂率转为下降（Table1）。而卵母细胞受精子激活和受精则不同,卵龄为15h ,卵母细胞的体外受精率为45．4％;随着卵龄的进一步增加,体外受精率则下降（Table2)。Fig.1显示：新排出的卵母细胞容易被精子激活而受精;卵龄较大的卵母细胞较易被酒精的人工刺激而激活。可能是卵母细胞从成熟到老化过程中,细胞的结构、功能及对外界刺激的敏感状态都在发生一些规律性的变化,而激活和受精的机制不完全,还不能精对卵龄的要求要严格。     

猪胞浆内单精子注射研究进展

胞浆内单精子注射将体外受精和显微操作技术相结合,已成功应用于哺乳动物受精机理研究,家畜性控胚胎生产,动物转基因等领域。综述了猪胞浆内单精子注射的主要影响因素,包括卵母细胞体外成熟和预处理、精子的选择和处理、注射卵的人工激活以及操作技术的改进,为进一步提高猪胞浆内单精子注射的效率以及该技术的应用提供参考。     

小鼠精子注入兔卵母细胞受精研究

利用显微操作仪将小鼠精子注入家兔卵母细胞的胞质内和透明带下,对鼠兔异种精卵互作和异种受精胚胎的发育进行了研究,并对注射精子的数量及卵的体外成熟时间等影响鼠兔异种显微受精的因素进行了探讨,结果如下:(1)将小鼠精子分别注入兔卵胞质内和透明带下,均能激活兔卵母细胞,导致精核解聚和原核形成;(2)小鼠精子注入兔卵胞质内和透明带下受精,杂种胚胎体外培养能发育到8-细胞期;(3)鼠兔异种受精4-细胞胚胎染色体标本制备观察结果表明,它们为正常二倍体;(4)鼠兔异种受精4-细胞胚胎的超微结构观察结果表明,它们极近似兔正常4-细胞胚胎的超微结构;(5)将小鼠精子注入兔卵透明带下,注射5—10个精子组卵的受精率(32.4%)和卵裂率(16.2%)均高于注射单个精子组的,但二组间差异不显著(P>0.05);DM 15%NCS液中体外成熟培养11—12h兔卵透明带下注入1—2个小鼠精子后的受精率(42.3%)和卵裂率(30.8%)均高于体外成熟培养24—25h组的,但二组间差异未达到显著水平(P>0.05)。     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。     

不同卵龄小鼠卵母细胞激活和受精的比较研究(英文)

本实验比较了不同卵龄的小鼠卵母细胞受酒精人工刺激后的激活率和体外受精率,以探讨卵母细胞激活和受精的机制。向ＮＩＨ雌鼠腹腔注射孕马血清促性腺激素（ＰＭＳＧ）７．５单位,４８小时后注射人绒毛膜促性激素（ＨＣＧ）７．５单位,于不同时间杀小鼠,取卵母细胞与卵丘细胞的复合体（ＯＣＣ）。从注射ＨＣＧ后到取ＯＣＣ的时间视为卵母细胞的卵龄。将ＯＣＣ置于含８％酒精的Ｍ２中７分钟,再在Ｍ１６中培养５小时后,用０．３ｍｇ／ｍＬ的透明质酸酶去卵丘细胞。卵母细胞形成原核或速即卵裂为激活的标志。将ＯＣＣ加入已获能的精子悬液中,５小时后将从卵丘细胞中释放出来的卵母细胞转移到Ｍ１６中,次日发生卵裂为卵母细胞体外受精和激活的标志。小鼠卵母细胞卵龄为２０ｈ,其激活率为８１．６％,速即卵裂率为４８．０％;而卵龄进一步增加到２４ｈ,激活率和卵裂率转为下降（Ｔａｂｌｅ１）。而卵母细胞受精子激活和受精则不同,卵龄为１５ｈ,卵母细胞的体外受精率为４５．４％;随着卵龄的进一步增加,体外受精率则下降（Ｔａｂｌｅ２）。Ｆｉｇ．１显示：新排出的卵母细胞容易被精子激活而受精;卵龄较大的卵母细胞较易被酒精的人工刺激而激活。可能是卵母细胞从成熟到老化过程中,细胞的结     

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     

卵母细胞激活

卵母细胞可被精子、化学物质和电脉冲等激活,激活后,胞质内发生Ca z 振荡、皮质颗粒释放、pH值增长以及成熟促进因子和有丝分裂原活化蛋白激酶失活等一系列重要事件.卵母细胞激活是人工构建孤雌胚胎和克隆胚胎的必需步骤.     

卵母细胞激活

卵母细胞可被精子、化学物质和电脉冲等激活,激活后,胞质内发生Ca2＋振荡、皮质颗粒释放、pH值增长以及成熟促进因子和有丝分裂原活化蛋白激酶失活等一系列重要事件。卵母细胞激活是人工构建孤雌胚胎和克隆胚胎的必需步骤。     

THE MECHANISM OF SPERM HEAD TRANSFORMATION DURING MOUSE OOCYTE FERTILIZATION IN VITRO

用Ｈｏｅｃｈｓｔ３３３４２标记及氨银反应的方法在光镜和电镜水平上研究不同发育阶段的小鼠卵母细胞转化精核的能力。生发泡期卵母细胞质不能诱发精子组蛋白替代鱼精蛋白及精核解聚。生发泡破裂后,卵母细胞获得使精核解聚的能力,但直到卵母细胞完成成熟前,雄原核均不能形成。进一步研究表明,卵母细胞成熟过程中持续的蛋白质合成是雄原核形成所必需的,鱼精蛋白质磷酸化是精核解聚中的关键步骤。     

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.     

Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.     

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n = 35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome. Mol. Reprod. Dev. 51:436–444, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull,boar, and ram sperm microinjected into hamster oocytes

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Rhesus macaque blastocysts resulting from intracytoplasmic sperm injection of vacuum‐dried spermatozoa*

Background Sperm desiccation is an attractive approach for sperm preservation. In this study, we examined the feasibility and efficiency of intracytoplasmic sperm injection using vacuum‐dried rhesus macaque sperm in CZB medium supplemented with 10% fetal bovine serum. Methods A total of 109 MII oocytes were injected with 69 fresh ejaculated sperm and 40 vacuum‐dried sperm. Results Cleavage occurred in 97% of oocytes injected with fresh, motile sperm and in 88% of oocytes injected with vacuum‐dried sperm. Of the cleaved oocytes, 68% fresh sperm‐injected oocytes and 74% of dried sperm‐injected oocytes developed to the compact morula stage. Blastocyst development was comparable between fresh‐injected (16%) and vacuum‐dried‐injected (17%) oocytes. Differences between treatment groups were not significant. Transmission electron microscopic observation of the blastocysts indicated no detectable differences between fresh sperm and dried sperm‐derived embryos. Conclusions We conclude that vacuum‐dried rhesus macaque sperm are capable of inducing fertilization and development of pre‐implantation embryos when sperm were dried under vacuum and microinjected into normal viable oocytes.     

In vitro fertilization of hamster and human oocytes by microinjection of human sperm

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 10⁶ sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.     

Sperm-derived activating ability does not persist in mouse oocytes inseminated during in vitro maturation

Activity of the sperm-derived oocyte-activating factor persists in zygotes and can be detected by a fusion with metaphase II (MII) oocytes leading to the activation of the hybrids. We have shown, that in the great majority of oocytes inseminated 1-2 hr after germinal vesicle breakdown (GVBD) the sperm-derived activating ability was eliminated. Only few hybrids produced by fusion of MII oocytes with oocytes inseminated during in vitro maturation (M x IVM-P + sperm hybrids) underwent activation, whereas almost all of MII oocyte x zygote hybrids entered interphase. However, frequency of activation of M x IVM-P + sperm hybrids was higher than that of control hybrids, which were obtained by fusion of MII oocytes with oocytes uninseminated during in vitro maturation. Although the difference was not statistically significant, it suggested that in a certain number of oocytes inseminated after GVBD the sperm-derived oocyte-activating factor remained partially active. This was confirmed by our observation that several oocytes, which were inseminated during in vitro maturation and managed to accomplish MII, underwent activation and formed pronuclei when examined 25-26 hr after the beginning of maturation. We have also demonstrated that parthenogenotes, could acquire the sperm-derived activity, as a consequence of sperm injection. MII oocytes were fused with parthenogenotes inseminated by ICSI and all hybrids underwent activation. This result indicated that the ability to induce activation in hybrid, was sperm-derived.